Methods and systems for inferring bovine traits

ABSTRACT

Methods, compositions, and systems are provided for managing bovine subjects in order to maximize their individual potential performance and edible meat value, and to maximize profits obtained in marketing the bovine subjects. The methods and systems draw an inference of a trait of a bovine subject by determining the nucleotide occurrence of at least one bovine SNP that is identified herein as being associated with the trait. The inference is used in methods of the present invention to establish the economic value of a bovine subject, to improve profits related to selling beef from a bovine subject; to manage bovine subjects, to sort bovine subjects; to improve the genetics of a bovine population by selecting and breeding of bovine subjects, to clone a bovine subject with a specific trait, to track meat or another commercial product of a bovine subject; and to diagnose a health condition of a bovine subject. Methods are also disclosed for identifying additional SNPs associated with a trait, by using the associated SNPs identified herein.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. §119(e)of U.S. Ser. No. 60/437,482, filed Dec. 31, 2002, the entire content ofwhich is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to gene association analyses and morespecifically to polymorphisms and associated traits of bovine species.

2. Background Information

Under the current standards established by the United States Departmentof Agriculture (USDA), beef from bulls, steers, and heifers isclassified into eight different quality grades. Beginning with thehighest and continuing to the lowest, the eight quality grades areprime, choice, select, standard, commercial, utility, cutter and canner.The characteristics which are used to classify beef include age, color,texture, firmness, and marbling, a term which is used to describe therelative amount of intramuscular fat of the beef. Well-marbled beef frombulls, steers, and heifers, i.e., beef that contains substantial amountsof intramuscular fat relative to muscle, tends to be classified as primeor choice; whereas, beef that is not marbled tends to be classified asselect. Beef of a higher quality grade is typically sold at higherprices than a lower grade beef. For example, beef that is classified as“prime” or “choice,” typically, is sold at higher prices than beef thatis classified into the lower quality grades.

Classification of beef into different quality grades occurs at thepacking facility and involves visual inspection of the ribeye on a beefcarcass that has been cut between the 12th and 13th rib prior tograding. However, the visual appraisal of a beef carcass cannot occuruntil the animal is harvested. Ultrasound can be used to give anindication of marbling prior to slaughter, but accuracy is low ifultrasound is done at a time significantly prior to harvest.

Currently there are no cost effective methods for identifying livecattle that give accurate prediction of the genetic potential to producebeef that is well-marbled. Such information could be used by feedlotoperators to identify animals for purchase prior to finishing, toidentify animals under contract for one or more premium programsadministered by a packer, by feedlot managers to make managementdecisions regarding individual animals within a lot (including nutritionprograms and sale dates), by cow-calf producers in marketing theiranimals to various feedlots or in making decisions regarding whichanimals will be sold on various carcass evaluation grids. Suchinformation could also be used to identify cattle that are goodcandidates for breeding. Thus, it is desirable to have a method whichcan be used to assess the beef marbling potential of live cattle,particularly young cattle well in advance of the arrival of the animalat the packing house.

Another characteristic of beef that is desired by consumers istenderness of the cooked product. Currently there are no procedures foridentifying live animals whose beef, if cooked properly, would betender. Currently, there are two types of procedures which are used byresearchers to assess the tenderness of meat samples after they havebeen aged and subsequently cooked. The first involves a subjectiveanalysis by a panel of trained testers. The second type is characterizedby methods used to cut or shear meat samples that have been removed froman animal and aged. One such method is the Warner-Bratzler shear forceprocedure which involves an instrumental measurement of the forcerequired to shear core samples of whole muscle after cooking. Neither ofthese procedures can be used to any practical effect in a fabricationsetting as the need to age product prior to testing would lead tomaintenance of inventory of fabricated product that would be costprohibitive. Consequently, the methods are used at research facilitiesbut not at packing plants. Accordingly, it is desirable to have newmethods which can be used to identify carcasses and live cattle thathave the potential to provide beef that, if cooked properly, will betender.

It has been difficult for the livestock industry to combine genetics forred meat yield and marbling and/or tenderness. In fact, conventionalmeasurement techniques indicate that marbling and red meat yield tend tobe antagonistic. Hence, there is a need for tools that identify superiorgenetic potential for the combination of red meat yield, tenderness andmarbling. Another trait of interest is live cattle growth rate (averagedaily gain). Currently, cattle producers do not have tools to identifyanimals with superior genetic potential for rapid growth prior topurchase. In addition, there are no methods currently available toidentify animals which combine capability for superior growth rate withdesirable carcass characteristics.

While many methods of measurement and selection of cattle in feedlotshave been tried, both visual and automated, such as ultrasound, nonehave been successful in accomplishing the desired end result. That endresult is the ability to identify and select cattle with superiorgenetic potential for desirable characteristics and then manage a givenanimal with known genetic potential for shipment at the optimum time,considering the animal's condition, performance and market factors, theability to grow the animal to its optimum individual potential ofphysical and economic performance, and the ability to record andpreserve each animal's performance history in the feedlot and carcassdata from the packing plant for use in cultivating and managing currentand future animals for meat production. The beef industry is extremelyconcerned with its decreasing market share relative to pork and poultry.Yet to date, it has been unable to devise a system or method toaccomplish on a large scale what is needed to manage the currentdiversity of cattle (i.e. least about 100 different breeds andco-mingled breeds) to improve the beef product quality and uniformityfast enough to remain competitive in the race for the consumer dollarspent on meat.

Modern day breeding programs in animal agriculture originated fromfundamental observations made upon the first domestication of animals.Early humans observed differences in a broad range of characteristicsbetween the offspring produced by mating different parents and they tookadvantage of this observation by only mating individuals thatdemonstrated the most desirable characteristics. By following thisstrategy for several generations our ancestors were able to createpopulations of animals that exhibited only desirable traits that bestfit their needs. This strategy, called selective mating or selectivebreeding, is based on identifying the best progeny from one generationand making them the parents for the next generation. Selective breedingresults in the development of individuals that are superior for one ormore traits and is the backbone for modern day genetic improvementprograms in animal agriculture.

Through the utilization of selective breeding strategies geneticistshave been able to define the fundamental genetic parameters thatinfluence the expression of traits. Breeding experiments revealed thatsome traits, like coat color, were expressed in a qualitative manner andcould be easily passed onto the next generation while other traits, likegrowth rate or adult size, were expressed in a quantitative fashion andonly small progress could be made at each generation. Subsequentresearch in the field of molecular genetics has now revealed thatqualitative trait effects are caused by the action of a single genewhile quantitative traits are caused by the action and interaction ofmany different genes.

In addition to contributions of genetics, it has been determined thatgenetic source alone did not account for all of the differences observedamong groups of closely related individuals and that environment andmanagement also played a role in determining the expression of specifictraits. In order to account for all of the differences observed betweenindividuals for a specific trait geneticists developed the equation; P(phenotype or overall trait expression)=G (genetic contribution fromparents)+E (contribution from the environment). Geneticists observedthat some traits respond better to selection than others due tointrinsic differences in G and E and developed scientific methods fordetermining the genetic contribution, or heritability, for a number ofunique traits. For any given trait a higher heritability indicates moreof the total variation is accounted for by the genetic source and afaster response to selection can be achieved. The parameters that governdifferences in the expression of specific traits between individuals asdefined above have been used for decades to make genetic improvement inanimal agriculture production. Utilization of these parameters in a“Classical Breeding Program” provides breeders with a set of tools toevaluate the genetic makeup of different individuals within a populationand to make steady progress in improving the expression of traits thathave economic significance to the commercial production of livestockspecies.

The primary objective of any genetic improvement program is to ascertainthe genetic potential of individuals for a broad range of economicallyimportant traits at a very early age. While the classical breedingapproach has produced steady genetic improvement in livestock species itis limited by the fact that accurate prediction of an individual'sgenetic potential can only be achieved when the animal reaches adulthood(fertility and production traits) or is harvested (meat quality traits).This is particularly problematic for meat animals since harvestedanimals obviously cannot enter the breeding pool. Furthermore, it isdifficult to utilize the classical breeding approach for traits that aredifficult (disease resistance) or costly (meat tenderness) to measure.

To overcome the previous problems with the classical breeding approachanimal breeders and geneticists turned to the new fields of moleculargenetics and genomics. These disciplines offered the promise that theunderlying genes responsible for genetic variation of important traitscould be identified. Targeted research programs were initiated toascertain the location and functional differences of specific genes thatcontribute to genetic variation for defined traits. The primary goal ofmolecular breeding programs in livestock species is to develop geneticassays for economically important traits that can be tested onindividual animals at an early age, can be used for traits that aredifficult to measure, that provide an accurate estimate of an animalsgenetic potential for expression of the trait, and account for a largeproportion of the total genetic variation observed for the trait incommercial populations.

To date, three different experimental approaches have been utilized toidentify genes that effect economically important traits in livestockspecies: Candidate Gene Approach, Differential Gene Expression Approach,and Within Family Quantitative Trait Loci (QTL) Linkage Approach.Limited success has been achieved for each of these methods inidentifying genes that contribute to genetic variation for definedtraits. However, each method also has limitations, as the primaryobjectives of the molecular breeding approach described above have notbeen achieved. Accordingly, a need exists for methods that assist in adetermination of the genetic potential of individuals for a broad rangeof economically important traits at a very early age. A description ofeach of the experimental approaches attempted thus far, and thelimitations for each is outlined below:

In the candidate gene approach a specific gene or set of genes istargeted based on the hypothesis they may have an effect on a particulartrait. The hypothesis is developed based on existing information ofbiochemical pathways and the function of the gene in another species,most often human or mouse where substantial gene characterization hasbeen performed. The known sequence of the human or mouse gene is used tofish-out the gene in the target species. The DNA sequence of the gene inthe target species is determined by sequencing a large number ofindividuals and any sequence variation is cataloged. The sequencevariations are developed into diagnostic assays and genotyped against apopulation of animals where phenotypic variation for the targeted traitshas been characterized. The data set is analyzed to determine ifstatistically significant associations exist between specific sequencevariants and expression of the trait.

The candidate gene approach has been successful in identifying genes andsequence variants that have an effect on a particular trait. However,this approach does have limitations and is analogous to finding aneedle-in-the-haystack. With over 30,000 genes characterized in humansand mouse as a result of the whole genome sequence the first difficultyis identifying a gene that will actually contribute to genetic variationfor a specific trait. Secondly, a large enough set of individuals mustbe sequenced to find the sequence variant that is responsible for or atleast highly associated with the effect. And finally, if an effect ispresent at all the population of animals screened must be large enoughto ensure statistically significant association of the effect. While itis feasible to meet all of these conditions to discover significantassociations the cost of this approach is high because it is a randommethod that cannot be targeted to genes that have the largest effect.

In the differential gene expression approach, differences in geneexpression are characterized for specific genes and in targeted tissueswith the hope of identifying genes that may be contributing to theobserved genetic variation for a particular trait. As in the CandidateGene Approach, targeted genes and tissues are chosen based on existinginformation of biochemical pathways and the functions of genes in otherspecies. Differential gene expression has been effective in identifyinggenes that are turned on or off by extreme differences in environment orby disease, but has been less successful in identifying genes thatcontribute to phenotypic variation in livestock production traits.Current technology platforms for detecting differences in geneexpression require large differences in gene expression, often up to a 2to 3 fold increase or decrease. Gene expression differences that mayaccount for genetic variation in livestock traits may be under thedetection threshold for existing gene expression technology.

Differential gene expression technology has been successfully used toelucidate biochemical pathways and to understand basic cellularfunctions but has not demonstrated any utility in the development ofdiagnostic assays to predict genetic potential of animals for specifictraits. Even if differential expression of a gene is observed and can bedirectly attributed to phenotypic variation for a trait there is noguarantee that a sequence variant can be found in the gene or that thesequence variant is responsible for the effect. In many situationssequence variants for differentially expressed genes do not associationwith the observed difference in phenotypes. This could be explained bythe action of other genes or gene products that regulate the expressionof the differentially expressed gene but are located elsewhere in thegenome.

In the within-family QTL linkage approach, small families of relatedindividuals are bred-up or assembled, DNA samples are taken from allindividuals in the population, phenotypic measurements for the targetedtraits are taken on the progeny and a set of polymorphic DNA markersthat span the genome are genotyped against the entire researchpopulation. The data set is then analyzed to determine if a particularmarker or a linked set of markers have specific allele(s) thatpredominately associate with the phenotypic variation observed in theprogeny from a specific parent or set of parents. A large number ofresearch reports claiming linkage between specific traits and markershave been published for a wide variety of traits and in severaldifferent livestock species.

Although the within family QTL linkage approach has resulted in a numberof reported linkages between targeted traits and specific markerlocations this approach does not result in the direct development ofdiagnostic assays that can predict an animals genetic potential for thetargeted trait. In practice, the research populations used for theseexperiments are very small, often only representing two or threedifferent sire families, and as such, they do not represent the broadpattern of genetic variation that is observed across commercial animalpopulations. These small research populations are also problematicbecause the QTL can only be identified when it is heterozygous for aparticular family group. Linkages between a marker and a trait aredetermined by allele frequency differences in the marker between progenyseparated into groups with high versus low expression for the trait.This implies that the QTL itself must be heterozygous in order to bedetected and the smaller the population the less likely it is to findQTLs in a heterozygous state. Furthermore, research populations designedto identify linkages in livestock species are usually half-sib designswhere it is only possible to measure the genetic variation contributedby the male side of the pedigree. Half-sib designs have limitedeffectiveness in discovering significant linkages because only one-halfof the genetic variation is accounted for in the analysis. Finally, theresearch populations are often comprised of animals and/or breed typesthat have extreme phenotypic differences for the targeted traits toinsure the discovery of markers that demonstrate linkage to the trait.These extreme phenotypic crosses do not represent mainstream industrybreeding practices and therefore, any reported linkage is suspectbecause it may only exist as an artifact within the research populationand may not actually be segregating in commercial animal breedingpopulations.

Another limitation of the within family QTL approach is the lack ofmarker density for the linkage map used in the study. Due to cost andgenotyping throughput issues all reported QTL linkage studies performedto date in livestock species have only used 100 to 200 total markers tocover the entire genome. With such a limited number of markers it isimpossible to pinpoint the exact location of the QTL on the chromosome.Linkage distances ranging from 3 to over 60 centi-Morgans are commonlyreported between the QTL and the linked marker(s). These broad linkagegroups can actually span an entire chromosome and contain thousands ofgenes that are possible candidates for the observed effect. Because ofthese large distances, recombination between homologous chromosomes doesnot allow the use of linked markers identified in research populationsto be used as predictors of genetic potential in commercial animalpopulations. Markers linked to QTLs can provide clues about thepotential location of genes that have effects for certain traits butsubstantial additional research and validation is required to accuratelypinpoint the location of the gene responsible for the effect and developdiagnostic assays to predict the expression of the trait.

In summary, three different experimental approaches have been used withlimited success to identify genes, chromosomal regions or DNA markersthat account for a large proportion of the genetic variation observed ineconomically important traits in livestock species. The results achievedfrom research programs utilizing these methods have not been widelyutilized to date because they do not account for enough of the totalgenetic variation to allow accurate prediction of an animal'sperformance for a specific trait. Furthermore, even when successfulthese approaches are only capable of identifying additive geneticcomponents while ignoring non-additive genetic components such asdominance (i.e. dominating trait of an allele of one gene over an alleleof a another gene) and epistasis (i.e. interaction between genes atdifferent loci) which are critical to the development of diagnosticsthat can be utilized by animal breeders to accurately predict geneticpotential for economically important traits in livestock species.

SUMMARY OF THE INVENTION

The present invention provides methods, systems, and compositions thatallow the identification and selection of cattle with superior geneticpotential for desirable characteristics. Accordingly, the presentinvention provides methods, compositions, and systems for managing,selecting and mating, breeding, and cloning cattle. These methods foridentification and monitoring of key characteristics of individualanimals and management of individual animals maximize their individualpotential performance and edible meat value. The methods of theinvention provide systems to collect, record and store such data byindividual animal identification so that it is usable to improve futureanimals bred by the producer and managed by the feedlot. The methods,compositions, and systems provided herein utilize information regardinggenetic diversity among cattle, particularly single nucleotidepolymorphisms (SNPs), and the effect of nucleotide occurrences of SNPson important traits.

The present invention further provides methods for selecting a givenanimal for shipment at the optimum time, considering the animal'sgenetic potential, performance and market factors, the ability to growthe animal to its optimum individual potential of physical and economicperformance, and the ability to record and preserve each animal'sperformance history in the feedlot and carcass data from the packingplant for use in cultivating and managing current and future animals formeat production. These methods allow management of the current diversityof cattle to improve the beef product quality and uniformity, thusimproving revenue generated from beef sales.

This invention allows the identification of animals that have superiortraits that can be used to identify parents of the next generationthrough selection. These methods can be imposed at the nucleus or elitebreeding level where the improved traits would, through time, flow tothe entire population of animals, or could be implemented at themultiplier or foundation parent level to sort parents into mostgenetically desirable. The optimum male and female parent can then beidentified to maximize the genetic components of dominance andepistasis, thus maximizing heterosis and hybrid vigor in the marketanimals.

In one embodiment, the present invention provides an isolatedpolynucleotide that includes at least 20 contiguous nucleotides of anyone of SEQ ID NOS:24493 to 64886, a polynucleotide at least 90%identical to the 20 contiguous nucleotide fragment, or a complementthereof, wherein the isolated polynucleotide includes a nucleotideoccurrence of a single nucleotide polymorphism (SNP) associated with atrait, wherein the SNP corresponds to position 300 of SEQ ID NOS:19473to 21982.

In another embodiment, the invention provides methods to draw aninference of a trait of a bovine subject by determining the nucleotideoccurrence of at least one bovine SNP that is determined using methodsdisclosed herein, to be associated with the trait. For example, theinference can be drawn by determining the nucleotide occurrence of atleast one SNP identified in Tables 1A and 1B (i.e. a SNP correspondingto position 300 of SEQ ID NOS:19473 to 21982). The inference can bedrawn regarding, for example, fat thickness, retail yield, marbling,tenderness, or average daily gain.

The inference is used in methods of the present invention for thefollowing aspects of the invention: to establish the economic value of abovine subject; to improve profits related to selling beef from a bovinesubject; to manage bovine subjects; to sort bovine subjects; to improvethe genetics of a bovine population by selecting and breeding of bovinesubjects; to clone a bovine subject with a specific trait, a combinationof traits, or a combination of SNP markers that predict a trait; totrack meat or another commercial product of a bovine subject; to certifyand brand a specific product based on known characteristics; and todiagnose a health condition of a bovine subject.

In another embodiment, the present invention provides a method foridentifying a bovine target sequence, such as a gene, associated with atrait, by identifying an open reading frame present in a target regionof the bovine genome, wherein the target region is located on the bovinegenome less than or equal to about 500,000 nucleotides of a singlenucleotide polymorphism (SNP) corresponding to position 300 of any oneof SEQ ID NOS:19473 to 21982, and analyzing the open reading frame todetermine whether it affects the trait, thereby identifying a bovinegene associated with the trait. In one aspect, the target region islocated within about 5000 nucleotides of a single nucleotidepolymorphism (SNP) corresponding to position 300 of any one of SEQ IDNOS:19473 to 21982.

In another embodiment, the present invention provides a method foridentifying a bovine single nucleotide polymorphism (SNP) associatedwith a trait, that includes identifying a test SNP in a target region ofa bovine genome, wherein the target region is less than or equal toabout 500,000 nucleotides of a SNP position corresponding to position300 of one of SEQ ID NOS:19473 to 21982, and identifying an associationof the test SNP to the trait, thereby identifying the test SNP asassociated with the trait. In certain aspects, the target regionincludes at least 20 contiguous nucleotides of SEQ ID NOS:24493 to64886. In another aspect, for example, the target region includes atleast 20 contiguous nucleotides of SEQ ID NOS:19473 to 21982. Thepresent invention also provides isolated polynucleotides that includethe identified SNPs.

DETAILED DESCRIPTION OF THE INVENTION

The specification hereby incorporates by reference in their entirety,the files contained on the two compact discs filed herewith. Two copiesof each of the two compact discs are filed herewith. The first compactdisc includes a file called “MMI1100-1 Table 1A.doc,” created Dec. 31,2003, which is 11299 kilobytes in size, and a file called “MMI1100-1Table 1B.doc,” created Dec. 31, 2003, which is 11266 kilobytes in size.The Second disc includes a sequence listing which is included in a filecalled “MMI1100-1 SEQUENCE LISTING.txt,” created Dec. 31, 2003, which is4770 kilobytes in size.

The compositions, methods, and systems of the invention are particularlywell suited for managing, selecting or mating bovine subjects. Theyallow for the ability to identify and monitor key characteristics ofindividual animals and manage those individual animals to maximize theirindividual potential performance and edible meat value. Therefore, themethods, systems, and compositions provided herein allow theidentification and selection of cattle with superior genetic potentialfor desirable characteristics.

The compositions, methods, and systems of the present invention areespecially well-suited for implementation in a feedlot environment. Theyallow for the ability to identify and monitor key characteristics ofindividual animals and manage those individual animals to maximize theirindividual potential performance and edible meat value. Furthermore, theinvention provides systems to collect, record and store such data byindividual animal identification so that it is usable to improve futureanimals bred by the producer and managed by the feedlot. The systems canutilize computer models to analyze information regarding nucleotideoccurrences of SNPs and their association with traits, to predict aneconomic value for a bovine subject.

Presently, feedlots contain pens which typically have a capacity ofabout 200 animals, and market to packers, pens of cattle that are fed toan average endpoint. The endpoint is calculated as a number of days onfeed estimated from biological type, sex, weight, and frame score.Animals are initially sorted to a pen based on the estimated number ofdays on feed and incoming group. However, sorting is done by a series ofsubjective and suboptimal parameters, as discussed herein. The cattleare fed to an endpoint in order to maximize the percentage of animalsfrom which Grade USDA Choice beef can be obtained at slaughter withoutdeveloping cattle that are too fat, and thus get discounted forinsufficient red meat yield. The present invention provides a method formaximizing a physical characteristic of a bovine subject, includingoptimizing the percentage of bovine subjects that produce Grade USDAChoice and Prime beef in the most efficient manner.

In one embodiment, the present invention provides an isolatedpolynucleotide that includes a fragment of at least 20 contiguousnucleotides of the bovine genome, or a complement thereof, wherein theisolated polynucleotide includes a nucleotide occurrence of a singlenucleotide polymorphism (SNP) associated with a trait, wherein the SNPis in disequilibrium with a SNP corresponding to position 300 of any oneof SEQ ID NOS:19473 to 21982. In certain aspects, the polynucleotide islocated about 500,000 or less nucleotides from position 300 of SEQ IDNOS:19473 to 21982 on the bovine genome. As disclosed in the Examplesherein, the linkage disequilibrium for cattle is about 500,000nucleotides. Therefore, it is expected that other SNPs can be identifiedthat are associated with the same traits based on the fact that theseother SNPs are located less than or equal to about 500,000 nucleotidesof the identified associated SNP on the bovine genome. In certainaspects, the polynucleotide is from an Angus, Charolais, Limousin,Hereford, Brahman, Simmental or Gelbvieh bovine subject.

The attached sequence listing provides sequences of contigs (SEQ IDNOS:24493 to 64886) generated from the bovine genome. It will beunderstood that contigs can be aligned such that SNPs that are onseparate contigs, but are located within 500,000 nucleotides on thebovine genome, can be identified. For example, alignment of contigs anddetermination of distance between contigs provided herein, can beaccomplished by using the sequence information of the human genome as ascaffold. Tables 1A and 113 (filed herewith on the compact disc), listscontigs that are “nearby” (i.e. within 500,000 nucleotides on the bovinegenome) an associated SNP.

In certain aspects, the isolated polynucleotide includes a nucleotidecorresponding to an associated SNP. Accordingly, in these aspects theisolated polynucleotide includes a nucleotide corresponding to position300 of any one of SEQ ID NOS:19473 to 21982.

In another aspect, the present invention provides an isolatedpolynucleotide that includes a polynucleotide that is at least 20nucleotides in length and is at least 90% identical to a fragment of atleast 20 contiguous nucleotides of a bovine genome; or a complementthereof, wherein the fragment of at least 20 contiguous nucleotides ofthe bovine genome comprises a nucleotide occurrence of a singlenucleotide polymorphism (SNP) associated with a trait, wherein the SNPis about 500,000 or less nucleotides from position 300 of any one of SEQID NOS:19473 to 21982. In certain aspects, for example, thepolynucleotide is at least 90% identical to a fragment of at least 10,15, 20, 25, 50, or 100 contiguous nucleotides of SEQ ID NOS:19473 to21982. In certain aspects, the polynucleotide comprises position 300 ofSEQ ID NOS:19473 to 21982.

As used herein, “about” means within ten percent of a value. Forexample, “about 100” would mean a value between 90 and 110.

In certain aspects, the isolated polynucleotide includes a fragment ofat least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 250, 500,1000, 5000, 10,000, 25,000, 50,000, 100,000, 125,000, 250,000 or 500,000nucleotides in length. Furthermore, in certain examples, the isolatedpolynucleotide includes a fragment of at least 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 100, 200, 250, 500, 1000, 5000, or 9549 contiguousnucleotides of any one of SEQ ID NOS:24493 to 64886. In another aspect,the isolated polynucleotide is at least 65, 70, 75, 80, 85, 90, 95, 96,97, 98, 99, or 99.5% identical to the recited sequences, for example. Inanother aspect, the isolated nucleotide includes region that iscontiguous with a region of any one of SEQ ID NOS:19473 to 21982 thatincludes position 300. In certain aspects, the isolated polynucleotideconsists of any one of SEQ ID NOS:19473 to 21982. In other aspects, theisolated polynucleotide consists of any one of SEQ ID NOS:21983 to24492.

The polynucleotide or an oligonucleotide of the invention can furtherinclude a detectable label. For example, the detectable label can beassociated with the polynucleotide at a position corresponding toposition 300 of any one of SEQ ID NOS:19473 to 21982. As discussed inmore detail herein, the labeled polynucleotide can be generated, forexample, during a microsequencing reaction, such as SNP-IT™ reaction.

Detectable labeling of a polynucleotide or oligonucleotide is well knownin the art. Particular non-limiting examples of detectable labelsinclude chemiluminescent labels, fluorescent labels, radiolabels,enzymes, haptens, or even unique oligonucleotide sequences.

In another embodiment, the present invention provides an isolated vectorthat includes a polynucleotide disclosed hereinabove. The term “vector”refers to a plasmid, virus or other vehicle known in the art that hasbeen manipulated by insertion or incorporation of a nucleic acidsequence.

Methods that are well known in the art can be used to construct vectors,including in vitro recombinant DNA techniques, synthetic techniques, andin vivo recombination/genetic techniques (See, for example, thetechniques described in Maniatis et al. 1989 Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y., incorporatedherein in its entirety by reference).

In another aspect, the present invention provides an isolated cell thatincludes the vector. The cell can be prokaryotic or eukaryotic.Techniques for incorporated vectors into prokaryotic and eukaryoticcells are well known in the art. In certain aspects, the cells arebovine cells. In other aspects, the cells are bacterial cells. In stillother aspects, the cells are human cells.

In another aspect, the present invention provides a primer pair thatbinds to a first target region and a second target region of SEQ IDNOS:24493 to 64886, wherein the first primer of the primer pair and asecond primer of the primer pair are at least 10 nucleotides in lengthand bind opposite strands of the target region located within 3000nucleotides of a position corresponding to position 300 of any one ofSEQ ID NOS:19473 to 21982, and prime polynucleotide synthesis from thetarget region in opposite directions across position 300. In anotherembodiment, provided herein is a primer pair that binds to a firsttarget region and a second target region of SEQ ID NOS:19473 to 21982,wherein a first primer of the primer pair and a second primer of theprimer pair are at least 10 nucleotides in length and bind oppositestrands of the target region, and prime polynucleotide synthesis fromthe target region in opposite directions across position 300 of SEQ IDNOS:19473 to 21982. In certain aspects, the target region is within SEQID NOS:19473 to 21982.

In another embodiment, the present invention provides an isolatedoligonucleotide that selectively binds to a target polynucleotide thatincludes at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 300,500, 1000, 1500, 2000, 2500, or 3000 nucleotides, for example, of SEQ IDNOS:24493 to 64886, wherein the terminal nucleotide corresponds toposition 300 of any one of SEQ ED NOS:19473 to 21982. In certainaspects, the isolated oligonucleotide includes at least 5 nucleotides ofSEQ ID NO:SNP1 to SNP4000. In certain aspects, the isolatedoligonucleotide is complementary to the nucleotide or a complementthereof, at position 299 or 300 of any one of SEQ ID NOS:19473 to 21982.

In another embodiment, the present invention provides an oligonucleotidethat binds to any one of SEQ ID NOS:19473 to 21982, wherein theoligonucleotide is between 10 and 50 nucleotides in length, and whereinthe oligonucleotide comprises at least 10 contiguous nucleotides of SEQID NOS:21983 to 24492. In certain aspects, for example, theoligonucleotide is at least 15 nucleotides in length. In certainexamples, the oligonucleotide binds to a region that includes position300 of any one of SEQ ID NOS:19473 to 21982. In other examples, theoligonucleotide includes at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24or 25 nucleotides of any one of SEQ ID NOS:21983 to 24492. The isolatedoligonucleotide can be any one of SEQ ID NOS:21983 to 24492.

In another embodiment, the present invention provides an isolatedoligonucleotide that includes 10 nucleotides, that selectively binds toa target polynucleotide of any one of SEQ ID NOS:19473 to 21982, whereina terminal nucleotide of the isolated oligonucleotide binds to position298, 299, 300, 301, or 302 of any one of SEQ ID NOS:19473 to 21982. Theoligonucleotide can be, for example, 10, 15, 20, 25, 50, or 100nucleotides in length. In certain aspects, the terminal nucleotide bindsto position 300 of any one of SEQ ID NOS:19473 to 21982.

In another embodiment, the present invention provides an isolatedoligonucleotide pair effective for determining a nucleotide occurrenceat a single nucleotide polymorphism (SNP) corresponding to position 300of any one of SEQ ID NOS:19473 to 21982, wherein each isolatedoligonucleotide comprises at least 5 nucleotides from SEQ ID NOS:19473to 21982 and wherein the terminal nucleotide of each oligonucleotidepair is complementary to a different nucleotide at position 300 of anyone of SEQ ID NOS:19473 to 21982 or a complement thereof. In certainaspects, the specific binding pair member is a substrate for a primerextension reaction.

In another embodiment, the invention provides a method for drawing aninference regarding a trait of a bovine subject by determining thenucleotide occurrence of at least one bovine SNP that is associated withthe trait. A SNP is associated with a trait when at least one nucleotideoccurrence of the SNP occurs more frequently in subjects with a certaincharacteristic of the trait in a statistically significant manner, forexample with greater than 80%, 85%, 90%, 95%, or 99% confidence.Therefore, in certain aspects, the methods include identifying whetherthe nucleotide occurrence is a bovine SNP allele identified herein asassociated with a trait. A bovine “SNP allele” is a nucleotideoccurrence of a SNP within a population of bovine animals. Theinference, in certain aspects, is drawn by determining the nucleotideoccurrence of one or more SNPs corresponding to position 300 of SEQ IDNOS:19473 to 21982. These SNPs are referred to herein as “associatedSNPs.” The inference can be drawn regarding a variety of traits asdiscussed herein, such as, for example, fat thickness, retail yield,marbling, tenderness, or average daily gain. In certain aspects, thebovine subject is an Angus, Charolais, Limousin, Hereford, Brahman,Simmental or Gelbvieh bovine subject.

As illustrated in the Example provided herein, a high density SNP map ofthe bovine genome was constructed and analyzed for the presence of SNPsthat are associated with a trait at a confidence level of 0.01 orgreater. The identified SNPs are referred to herein as “SNPs that areassociated with a trait” or “associated SNPs.” The predictive value ofthe associated SNPs allow a determination of the genetic potential of abovine animal to express multiple economically important traits, termedthe molecular breeding and selection value. This information is utilizedto enhance the efficiency and accuracy of breeding, sorting and cloningof animals.

The analysis disclosed in the Examples herein, utilized methods of thepresent invention, to generate a high-density genetic map of the bovinegenome based on single nucleotide polymorphic (SNP) markers. Thehigh-density genetic map was created through a whole genome sequence ofthe bovine genome using the shotgun sequencing approach as described byVenter, J. C, et al., (Science 291:1304-1351 (2001)). Shotgun sequencingwas performed with four different bovine individuals that representdifferent breed types. Upon whole genome assembly of the sequencedfragments all sequence variants were identified and cataloged. Sequencevariants that differ by a single nucleotide became candidate SNP markersfor the high-density map. The relative position of each candidate SNPwithin the bovine genome was determined by using the assembled humangenome as scaffolding. Candidate SNPs were chosen based on theirlocations so that the map is evenly distributed across the bovinegenome. The genetic SNP map is evenly distributed where the averagegenetic distance between any two adjacent markers is 0.5 cM.

Furthermore, phenotypic data from 3791 bovine animals was collected froma three by three factorial feeding and carcass data collectionexperiment, comparing three biological types (English, Continental andBrahman crosses) within three different days on feed (early, optimum andlate). Animals were randomly assigned to treatment groups based onbiological type. All cattle entered the experiment within 90 kg of bodyweight. These groups were blocked across starting and harvest date.Blood samples were collected on each individual animal at the start ofthe feeding period and assigned an electronic ID that was matched to thecollection sample. At the completion of the feeding and harvest perioddata were compiled and analyzed for relevant statistical parameters.Statistically significant associations between specific SNPs andtargeted traits were identified by methods disclosed herein forutilizing a high-density genetic SNP map in the performance of wholegenome association studies in bovine animals. Using methods and resultsprovided herein, the effect of the associated SNP on the target traitthrough allele frequency differences in the SNP was determined.Furthermore, as disclosed herein, SNPs that are adjacent to or in closeproximity to some of the associated SNPs were identified that areassociated with the same traits as an associated SNP.

As discussed in detail in the attached Examples, DNA samples were pooledfrom bovine subjects that represent high and low phenotypic extremes forthe expression of a target trait in a population of bovine animals (e.g.high fat). The traits selected for analysis were marbling, tenderness,fat thickness, yield, and daily gain. A total of 2510 SNPs wereidentified that are associated with these traits (Tables 1A and 1B).

Tables 1A and 1B, both of which are filed herewith on a compact disc,disclose the SNPs identified by the analysis, and provide the SNP namesfor the SNPs corresponding to position 300 of SEQ ID NOS:19473 to 21982.The sequences disclosed in SEQ ID NOS:SNP1 to SNP4000 are regions fromwhich amplicons were generated. Table 1B also indicates the location ofthe amplicon-generating regions within a larger bovine genomic sequencecontig (SEQ ID NOS:24493 to 64886) (See column 2 of Table 1B, labeled“In Sequence,” which lists a contig name (e.g., “19866880525139”) andpositions (e.g. “923-1522”) within the contig of an amplicon whichincludes the SNP at position 300. A sequence identifier for the amplicon(SEQ ID NOS:19473-21982) is listed in Table 1A. Furthermore, Tables 1Aand 1B identify the nucleotide occurrences that have been detected foreach of these SNPs, and identifies traits that have been identified tobe associated with these SNPs using methods disclosed herein. All of theSNPs listed in Tables 1A and 1B were associated with the respectivetrait(s) with a confidence level of 0.01, or higher confidence. Finally,Table 1A provides the sequence of an extension primer that was used todetermine the nucleotide occurrence of the SNPs (SEQ ID NOS:21983 to24492).

Each SNP in Tables 1A and 1B is characterized by the trait(s) found tobe in association: marbling, tenderness, fat thickness, daily gain andretail yield. For each of the five traits, “High” refers to animalsreaching the 90^(th) percentile of that phenotypic measurement based onnumeric ranking for the trait. “Low” refers to animals in the 10^(th)percentile or less of that phenotypic measurement based on the numericranking of the trait.

In certain aspects of the invention directed at methods for inferringtraits such as the traits listed in Tables 1A and 1B, nucleotideoccurrences are determined for one or more associated SNPs. Therefore,in one aspect, for example, the method is used to infer fat thickness,by determining a nucleotide occurrence of at least one SNP correspondingto the SNPs indicated in Tables 1A and 1B as associated with fatthickness. For this aspect, as a non-limiting example, a nucleotideoccurrence of the SNP at position 300 of SEQ ID NO:19473 can beidentified and compared to the nucleotide occurrences listed in Tables1A and 1B 1 for SEQ ID NO:19473. A thymidine residue at position 300 ofSEQ ID NO:19473 infers a higher likelihood that the bovine subject willproduce meat that has high tenderness. In addition, as a non-limitingexample, a nucleotide occurrence at position 300 of SEQ ID NO:19474 canbe determined and used alone or in combination with the nucleotideoccurrence at position 300 of SEQ ID NO:19473, to infer tenderness. Forexample, if position 300 of both SEQ ID NO:19473 and SEQ ID NO:19474 arethymidine residues, there is an even greater likelihood that the bovinesubject will produce meat that has high tenderness, than for eithernucleotide occurrence alone.

In another aspect, the method is used to infer retail yield, bydetermining a nucleotide occurrence of at least one SNP corresponding tothe SNPs indicated in Table 1A as associated with retail yield. Inanother aspect, the method is used to infer marbling by determining anucleotide occurrence of at least one SNP corresponding to the SNPsindicated in Table 1A as associated with marbling. In another aspect,the method is used to infer daily gain, by determining a nucleotideoccurrence of at least one SNP corresponding to the SNPs indicated inTable 1A as associated with daily gain.

For any trait, a “relatively high” characteristic, indicates greaterthan average, and a “relatively low” characteristic indicates less thanaverage. For example “relatively high marbling”, indicates more abundantmarbling than average marbling for a bovine population. Conversely,“relatively low marbling”, indicates less abundant marbling than averagemarbling for a bovine population. Furthermore, in certain aspects,methods of the present invention infer that a bovine subject has asignificant likelihood of having a value for a trait that is within, forexample, the 5th, 10th, 20th, 25th, 30th, 40th, 50th, 60th, 70th, 75th,80th, 90th, or 95th percentile of bovine subjects for a given trait. Forexample, a method presented herein can provide an inference that abovine subject has a significant likelihood of having a marbling valuethat is within the 10th percentile of marbling for a bovine population.SNP nucleotide occurrences listed in Tables 1A and 1B as associated witha “high” trait characteristic (e.g., high tenderness) are likely to beassociated with a value greater than a 50th percentile of the bovinepopulation for the relevant trait, and in certain aspects, in the atleast 90th percentile. SNP nucleotide occurrences listed in Tables 1Aand 1B as associated with a “low” trait characteristic (e.g., lowtenderness) are likely to be associated with a value less than a 50thpercentile of the bovine population for the relevant trait, and incertain aspects, less than or equal to the 10th percentile.

In one aspect, the methods of the invention can be utilized incombination with various hypermutable sequences, such as microsatellitenucleic acid sequences to infer traits of bovine subjects. As usedherein, the term “hypermutable” refers to a nucleic acid sequence thatis susceptible to instability, thus resulting in nucleic acidalterations. Such alterations include the deletion and addition ofnucleotides. The hypermutable sequences of the invention are most oftenmicrosatellite DNA sequences which, by definition, are small tandemrepeat DNA sequences. Thus, a combination of SNP analysis andmicrosatellite analysis may be used to infer a trait(s) of a bovinesubject.

In another embodiment, the present invention provides a method fordetermining a nucleotide occurrence of a polymorphism in a bovinesample, wherein polymorphism corresponds to position 300 of any one ofSEQ ID NOS:19473 to 21982. In one aspect, the nucleotide occurrence isdetermined by contacting a bovine polynucleotide in the sample with anoligonucleotide that binds to a target region of any one of SEQ IDNOS:24493 to 64886, wherein the target region comprises a positioncorresponding to position 300 of any one of SEQ ID NOS:19473 to 21982 orwherein the target region is within 3000 nucleotides of a nucleotidecorresponding to position 300 of any one of SEQ ID NOS:19473 to 21982,and determining the nucleotide occurrence of a single nucleotidepolymorphism (SNP) corresponding to position 300 of any one of SEQ IDNOS:19473 to 21982. The determination typically includes analyzingbinding of the oligonucleotide or detecting an amplification productgenerated using the oligonucleotide.

In certain aspects, the target region is within 3000, 2000, 1500, 1000,750, 500, 250, 200, 150, 100, 75, 50, 40, 30, 20 10, 9, 8, 8, 7, 6, 5,4, 3, 2, or 1 nucleotide of a nucleotide corresponding to position 300of any one of SEQ ID NOS:19473 to 21982, or the target region includesposition 300 of SEQ ID NOS:19473 to 21982. In certain aspects, thetarget region is any one of SEQ ID NOS:19473 to 21982.

In certain aspects, for example, the oligonucleotide binds to a targetsequence that includes one of the SNPs and the nucleotide occurrence isdetermined based on the binding of the oligonucleotide to the targetsequence. Methods for determining nucleotide occurrences at SNPs aredisclosed herein. Some of these methods utilize flanking primer pairs.Accordingly, in one aspect, the bovine polynucleotide is contacted witha primer pair, and the nucleotide occurrence is determined using anamplification product generated using the primer pair. One or both ofthe primers in the primer pair can include a detectable label.

In certain examples, the terminal nucleotide of the oligonucleotidebinds to the SNP position. For example, the terminal nucleotide of eacholigonucleotide pair can be complementary to a different nucleotide atposition 300 of any one of SEQ ID NOS:19473 to 21982 or a complementthereof. In certain aspects, one oligonucleotide is the oligonucleotideof any one of SEQ ID NOS:21983 to 24492.

In certain aspects, the method further includes managing at least one offood intake, diet composition, administration of feed additives orpharmacological treatments such as vaccines, antibiotics, hormones andother metabolic modifiers, age and weight at which diet changes orpharmacological treatments are imposed, days fed specific diets,castration, feeding methods and management, imposition of internal orexternal measurements and environment of the bovine subject based on theinferred trait. This management results in improved, and in someexamples, a maximization of physical characteristic of a bovine subject,for example to obtain a maximum amount of high grade beef from a bovinesubject, and/or to increase the chances of obtaining grade USDA Choiceor Prime beef, optimize tenderness, and/or maximize retail yield fromthe bovine subject taking into account the inputs required to reachthose endpoints.

The method can be used to discriminate among those animals where growthimplants, vitamin E, and other interventions could provide the greatestvalue. For example, animals that do not have the traits to reach highchoice or prime quality grades may be given growth implants until theend of the feeding period, thus maximizing feed efficiency while animalswith a propensity to marble may not be implanted at the final stages ofthe feeding period to ensure maximum fat deposition intramuscularly.

The method also allows a feedlot and processor to predict the qualityand yield grades of cattle in the system to optimize marketing of thefed animal or the product to meet target market specification. Themethod also provides information to the feedlot for purchase decisionsbased on the predicted economic returns from a specific supplier.Furthermore, The method allows the creation of integrated programsspanning breeders, producers, feedlots, packers and retailers.

Examples of feed additives include antibiotics, flavors and metabolicmodifiers. Information from SNPs could influence use of these additivesand other pharmacologic treatments depending on cattle genetic potentialand stage of growth relative to expected carcass composition. Examplesof feeding methods include ad-libitum versus restricted feeding, feedingin confined or non-confined conditions and number of feedings per day.Information from SNPs relative to cattle health, immune status or stressresponse could be used to influence choice of optimum feeding methodsfor individual cattle.

In another embodiment, methods are provided for selecting a given animalfor shipment at the optimum time, considering the animal's condition,performance and market factors, the ability to grow the animal to itsoptimum individual potential of physical and economic performance, andthe ability to record and preserve each animal's performance history inthe feedlot and carcass data from the packing plant for use incultivating and managing current and future animals for meat production.These methods allow management of the current diversity of cattle toimprove the beef product quality and uniformity, thus improving revenuegenerated from beef sales.

The methods can use a bioeconomic valuation method that establishes theeconomic value of a bovine subject, or a group of bovine subjects, suchas those in a pen, to optimize profits from beef production.Accordingly, in another embodiment, the present invention provides amethod for establishing the economic value of a bovine subject.According to the method, an inference is drawn regarding a trait of thebovine subject from a nucleic acid sample of the bovine subject. Theinference is drawn by a method that includes identifying nucleotideoccurrences for at least one single nucleotide polymorphism (SNP),wherein the nucleotide occurrence is associated with the trait, andwherein the trait affects the value of the bovine subject. Furthermore,the inference, in certain aspects, is drawn by determining thenucleotide occurrence of at least one SNP corresponding to position 300of SEQ ID NOS:19473 to 21982.

The method, in certain examples, includes identification of thecausative mutation influencing the trait directly or the determinationof 1 or more SNPs that are in linkage disequilibrium with the associatedtrait.

The method can include a determination of the nucleotide occurrence ofat least 2 SNPs. At least 2 SNPs can form all or a portion of ahaplotype, wherein the method identifies a haplotype allele that is inlinkage disequilibrium and thus associated with the trait. Furthermore,the method can include identifying a diploid pair of haplotype alleles.

A method according to this aspect of the invention can further includeusing traditional factors affecting the economic value of the bovinesubject in combination with the inference based on nucleotide occurrencedata to determine the economic value of the bovine subject.

As used herein, the term “at least one”, when used in reference to agene, SNP, haplotype, or the like, means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,etc., up to and including all of the haplotype alleles, genes, and/orSNPs of the bovine genome. Reference to “at least a second” gene, SNP,or the like, means two or more, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.,bovine genes, SNPs, or the like.

Polymorphisms are allelic variants that occur in a population that canbe a single nucleotide difference present at a locus, or can be aninsertion or deletion of one, a few or many consecutive nucleotides. Assuch, a single nucleotide polymorphism (SNP) is characterized by thepresence in a population of one or two, three or four nucleotides (i.e.,adenosine, cytosine, guanosine or thymidine), typically less than allfour nucleotides, at a particular locus in a genome such as the humangenome. It will be recognized that, while the methods of the inventionare exemplified primarily by the detection of SNPs, the disclosedmethods or others known in the art similarly can be used to identifyother types of bovine polymorphisms, which typically involve more thanone nucleotide.

The term “haplotypes” as used herein refers to groupings of two or moreSNPs that are physically present on the same chromosome which tend to beinherited together except when recombination occurs. The haplotypeprovides information regarding an allele of the gene, regulatory regionsor other genetic sequences affecting a trait The linkage disequilibriumand, thus, association of a SNP or a haplotype allele(s) and a bovinetrait can be strong enough to be detected using simple geneticapproaches, or can require more sophisticated statistical approaches tobe identified.

Numerous methods for identifying haplotype alleles in nucleic acidsamples are known in the art. In general, nucleic acid occurrences forthe individual SNPs are determined and then combined to identifyhaplotype alleles. There are several algorithms for haplotypereconstruction based on pedigree analysis. These are the MaximumLikelihood method ((Excofier, L., and Slatkin, M., Mol. Biol. Evol. 12:921-927 (1995)), the parsimony method created by Clark, A. G., Mol.Biol. Evol. 7: 111-122 (1990) and the phase reconstruction method ofStephens, M., et al., Am. J. Hum. Genet. 68:978-989, 2001, which isincorporated herein by reference) can be applied to the data generatedregarding individual nucleotide occurrences in SNP markers of thesubject, in order to determine alleles for each haplotype in a subject'sgenotype. Alternatively, haplotypes can also be determined directly, foreach pair of sites, by allele-specific PCR (Clark, A. G. et al., Am. J.Hum. Genet. 63: 595-612 (1998).

As used herein, the term “infer” or “inferring”, when used in referenceto a trait, means drawing a conclusion about a trait using a process ofanalyzing individually or in combination, nucleotide occurrence(s) ofone or more SNP(s), which can be part of one or more haplotypes, in anucleic acid sample of the subject, and comparing the individual orcombination of nucleotide occurrence(s) of the SNP(s) to knownrelationships of nucleotide occurrence(s) of the SNP(s) and the trait.As disclosed herein, the nucleotide occurrence(s) can be identifieddirectly by examining nucleic acid molecules, or indirectly by examininga polypeptide encoded by a particular gene where the polymorphism isassociated with an amino acid change in the encoded polypeptide.

Relationships between nucleotide occurrences of one or more SNPs orhaplotypes and a trait can be identified using known statisticalmethods. A statistical analysis result which shows an association of oneor more SNPs or haplotypes with a trait with at least 80%, 85%, 90%,95%, or 99%, or 95% confidence, or alternatively a probability ofinsignificance less than 0.05, can be used to identify SNPs andhaplotypes. These statistical tools may test for significance related toa null hypothesis that an on-test SNP allele or haplotype allele is notsignificantly different between groups with different traits. If thesignificance of this difference is low, it suggests the allele is notrelated to a trait.

As another example, associations between nucleotide occurrences of oneor more SNPs or haplotypes and a trait (i.e. selection of significantmarkers) can be identified using a two part analysis in the first part,DNA from animals at the extremes of a trait are pooled, and the allelefrequency of one or more SNPs or haplotypes for each tail of thedistribution is estimated. Alleles of SNPs and/or haplotypes that areapparently associated with extremes of a trait are identified and areused to construct a candidate SNP and/or haplotype set. Statisticalcut-offs are set relatively low to assure that significant SNPs and/orhaplotypes are not overlooked during the first part of the method.

During the second stage, individual animals are genotyped for thecandidate SNP and/or haplotype set. The second stage is set up toaccount for as much of the genetic variation as possible in a specifictrait without introducing substantial error. This is a balancing act ofthe prediction process. Some animals are predicted with high accuracyand others with low accuracy.

In diploid organisms such as bovines, somatic cells, which are diploid,include two alleles for each single-locus haplotype. As such, in somecases, the two alleles of a haplotype are referred to herein as agenotype or as a diploid pair, and the analysis of somatic cells,typically identifies the alleles for each copy of the haplotype. Methodsof the present invention can include identifying a diploid pair ofhaplotype alleles. These alleles can be identical (homozygous) or can bedifferent (heterozygous). Haplotypes that extend over multiple loci onthe same chromosome include up to 2 to the Nth power alleles where N isthe number of loci. It is beneficial to express polymorphisms in termsof multi-locus (i.e. multi SNP) haplotypes because haplotypes offerenhanced statistical power for genetic association studies. Multi-locushaplotypes can be precisely determined from diploid pairs when thediploid pairs include 0 or 1 heterozygous pairs, and N or N−1 homozygouspairs. When multi-locus haplotypes cannot be precisely determined, theycan sometimes be inferred by statistical methods. Methods of theinvention can include identifying multi-locus haplotypes, eitherprecisely determined, or inferred.

A sample useful for practicing a method of the invention can be anybiological sample of a subject, typically a bovine subject, thatcontains nucleic acid molecules, including portions of the genesequences to be examined, or corresponding encoded polypeptides,depending on the particular method. As such, the sample can be a cell,tissue or organ sample, or can be a sample of a biological material suchas a body fluid, for example blood, milk, semen, saliva, or can be hair,tissue, and the like. A nucleic acid sample useful for practicing amethod of the invention can be deoxyribonucleic (DNA) acid orribonucleic acids (RNA). The nucleic acid sample generally is adeoxyribonucleic acid sample, particularly genomic DNA or anamplification product thereof. However, where heteronuclear ribonucleicacid which includes unspliced mRNA precursor RNA molecules andnon-coding regulatory molecules such as RNA is available, a cDNA oramplification product thereof can be used.

Where each of the SNPs of the haplotype is present in a coding region ofa gene(s), the nucleic acid sample can be DNA or RNA, or productsderived therefrom, for example, amplification products. Furthermore,while the methods of the invention generally are exemplified withrespect to a nucleic acid sample, it will be recognized that particularhaplotype alleles can be in coding regions of a gene and can result inpolypeptides containing different amino acids at the positionscorresponding to the SNPs due to non-degenerate codon changes. As such,in another aspect, the methods of the invention can be practiced using asample containing polypeptides of the subject.

In one embodiment, DNA samples are collected and stored in a retrievablebarcode system, either automated or manual, that ties to a database.Collection practices include systems for collecting tissue, hair, mouthcells or blood samples from individual animals at the same time that eartags, electronic identification or other devices are attached orimplanted into the animal. Tissue collection devices can be integratedinto the tool used for placing the ear tag. Body fluid samples arecollected and can be stored on a membrane bound system. All methodscould be automatically uploaded into a primary database.

The sample is then analyzed on the premises or sent to a laboratorywhere a high-throughput genotyping system is used to analyze the sample.Traits are predicted in the field, in real-time, or in the laboratoryand forwarded electronically to a feedlot. The feedlot then uses thisinformation to sort and manage animals to maximize profitability andmarketing potential.

The present invention can also be used to provide information tobreeders to make breeding, mating, and or cloning decisions. Thisinvention can also be combined with traditional genetic evaluationmethods to improve selection, mating, or cloning strategies.

The subject of the present invention can be any bovine subject, forexample a bull, a cow, a calf, a steer, or a heifer or any bovine embryoor tissue, and includes all breeds of bovines. For methods of theinvention directed at sorting bovine subjects, managing bovine subjects,improving profits related to selling beef from a bovine subject, theanimal can be a young bovine subject ranging in ages from conception tothe time the animal is harvested and beef and other commercial productsobtained. The method of the present invention can be performed after theanimal is purchased and first enters the feedlot.

A “trait” is a characteristic of an organism that manifests itself in aphenotype. Many traits are the result of the expression of a singlegene, but some are polygenic (i.e., result from simultaneous expressionof more than one gene). A “phenotype” is an outward appearance or othervisible characteristic of an organism. Many different non-bovinelivestock traits can be inferred by methods of the present invention.Traits analyzed in methods of the present invention include, but are notlimited to, marbling, tenderness, quality grade, quality yield, musclecontent, fat thickness, feed efficiency, red meat yield, average dailyweight gain, disease resistance, disease susceptibility, feed intake,protein content, bone content, maintenance energy requirement, maturesize, amino acid profile, fatty acid profile, milk production, hidequality, susceptibility to the buller syndrome, stress susceptibilityand response, temperament, digestive capacity, production of calpain,calpastatin and myostatin, pattern of fat deposition, ribeye area,fertility, ovulation rate, conception rate, fertility, heat tolerance,environmental adaptability, robustness, susceptibility to infection withand shedding of pathogens such as E. Coli, Salmonella sp. and otherhuman pathogens.

Methods of the present invention can be used to infer more than onetrait. For example a method of the present invention can be used toinfer a population of traits. Accordingly, a method of the presentinvention can infer, for example, quality grade, muscle content, andfeed efficiency. This inference can be made using one SNP or apopulation or series of SNPs. Thus, a single SNP can be used to infermultiple traits; multiple SNPs can be used to infer multiple traits; ora single SNP can be used to infer a single trait. Where certain traitshave either positive or negative correlations to each other, the methodsallow identification of all SNPs that enhance or uncouple thecorrelation.

In another aspect, the present invention provides a method for improvingprofits related to selling beef from a bovine subject. The methodincludes drawing an inference regarding a trait of the bovine subjectfrom a nucleic acid sample of the bovine subject. The method istypically performed by a method that includes identifying a nucleotideoccurrence for at least one single nucleotide polymorphism (SNP),wherein the nucleotide occurrence is associated with the trait, andwherein the trait affects the value of the animal or its products.Furthermore, the method includes managing at least one of food intake,diet composition, administration of feed additives or pharmacologicaltreatments such as vaccines, antibiotics, hormones and other metabolicmodifiers, age and weight at which diet changes or pharmacologicaltreatments are imposed, days fed specific diets, castration, feedingmethods and management, imposition of internal or external measurementsand environment of the bovine subject based on the inferred trait. Thenat least one bovine commercial product, typically meat or milk, isobtained from the bovine subject.

Methods according to this aspect of the present invention can utilize abioeconomic model, such as a model that estimates the net value of oneor more bovine subjects based on one or more traits. By this method,traits of one, or a series of traits are inferred, for example, aninference regarding several characteristics of beef that will beobtained from the bovine subject. This inference is typically madebefore the bovine subjects enter the feedlot. The inferred traitinformation then can be entered into a model that uses the informationto estimate a value for the bovine subject, or beef from the bovinesubject, based on the traits. The model is typically a computer model.Values for the bovine subjects can be used to segregate the animals.Furthermore, various parameters that can be controlled duringmaintenance and growth of the bovine subjects can be input into themodel in order to affect the way the animals are raised in order toobtain maximum value for the bovine subject when it is harvested.

In certain embodiments, meat or milk can be obtained at a time pointthat is affected by the inferred trait and one or more of the foodintake, diet composition, and management of the bovine subject. Forexample, where the inferred trait of a bovine subject is high feedefficiency, which can be identified in quantitative or qualitativeterms, meat or milk can be obtained at a time point that is sooner thana time point for a bovine subject with low feed efficiency. As anotherexample, bovine subjects with different feed efficiencies can beseparated, and those with lower feed efficiencies can be implanted withgrowth promotants or fed metabolic partitioning agents in order tomaximize the profitability of a single bovine subject.

In another aspect, the present invention provides methods that alloweffective measurement and sorting of animals individually, accurate andcomplete record keeping of genotypes and traits or characteristics foreach animal, and production of an economic end point determination foreach animal using growth performance data. Accordingly, the presentinvention provides a method for sorting bovine subjects. The methodincludes inferring a trait for both a first bovine subject and a secondbovine subject from a nucleic acid sample of the first bovine subjectand the second bovine subject. The inference is made by a method thatincludes identifying the nucleotide occurrence of at least one singlenucleotide polymorphism (SNP), wherein the nucleotide occurrence isassociated with the trait. The method further includes sorting the firstbovine subject and the second bovine subject based on the inferredtrait.

The method can further include measuring a physical characteristic ofthe first bovine subject and the second bovine subject, and sorting thefirst bovine subject and the second bovine subject based on both theinferred trait and the measured physical characteristic. The physicalcharacteristic can be, for example, weight, breed, type or frame size,and can be measured using many methods known in the art, such as byusing ultrasound.

In another aspect, the present invention provides methods that useanalysis of bovine genetic variation to improve the genetics of thecattle population to produce animals with consistent desirablecharacteristics, such as animals that yield a high percentage of leanmeat and a low percentage of fat efficiently. Accordingly, in one aspectthe present invention provides a method for selection and breeding ofbovine subjects for a trait. The method includes inferring the geneticpotential for a trait or a series of traits in a group of bovinecandidates for use in breeding programs from a nucleic acid sample ofthe bovine candidates. The inference is made by a method that includesidentifying the nucleotide occurrence of at least one single nucleotidepolymorphism (SNP), wherein the nucleotide occurrence is associated withthe trait or traits. Individuals are then selected from the group ofcandidates with a desired performance for the trait or traits for use inbreeding programs. Progeny resulting from mating of selected parentswould contain the optimum combination of traits, thus creating anenduring genetic pattern and line of animals with specific traits. Theselines could be monitored for purity using the original SNP markers andcould be identified from the entire population of bovines and protectedfrom genetic theft.

In another aspect the present invention provides a method for cloning abovine subject with a specific trait or series of traits. The methodincludes identifying nucleotide occurrences of at least one or at leasttwo SNPs for the bovine subject, isolating a progenitor cell from thebovine subject, and generating a cloned bovine from the progenitor cell.The method can further include before identifying the nucleotideoccurrences, identifying the trait of the bovine subject, wherein thebovine subject has a desired trait and wherein the at least one or atleast two SNPs affect the trait.

Methods of cloning cattle are known in the art and can be used for thepresent invention. (See e.g., Bondioli, “Commercial cloning of cattle bynuclear transfer”, In: Symposium on Cloning Mammals by NuclearTransplantation, Seidel (ed), pp. 35-38, (1994); Willadsen, “Cloning ofsheep and cow embryos,” Genome, 31:956, (1989); Wilson et al.,“Comparison of birth weight and growth characteristics of bovine calvesproduced by nuclear transfer (cloning), embryo transfer and naturalmating”, Animal Reprod. Sci., 38:73-83, (1995); and Barnes et al.,“Embryo cloning in cattle: The use of in vitro matured oocytes”, J.Reprod. Fert., 97:317-323, (1993)). These methods include somatic cellcloning (See e.g., Enright B. P. et al., “Reproductive characteristicsof cloned heifers derived from adult somatic cells,” Biol. Reprod.,66:291-6 (2002); Bruggerhoff K., et al., “Bovine somatic cell nucleartransfer using recipient oocytes recovered by ovum pick-up: effect ofmaternal lineage of oocyte donors,” Biol. Reprod., 66:367-73 (2002);Wilmut, I., et al., “Somatic cell nuclear transfer,” Nature, 419:583(2002); Galli, C., et al., “Bovine embryo technologies,” Theriogenology,59:599 (2003); Heyman, Y., et al., “Novel approaches and hurdles tosomatic cloning in cattle,” Cloning Stem Cells, 4:47 (2002)).

Furthermore, methods have been reported for culturing bovine embryonicstem cells (See e.g., Saito, et al., “Bovine embryonic stem cell-likecell lines cultured over several passages,” Roux's Arch. Dev. Biol.,201:i34-i40, 1992). These cells can be used to produce tissues withpredetermined characteristics based on SNP information using the methodsof the present invention.

This invention identifies animals that have superior traits, predictedvery accurately, that can be used to identify parents of the nextgeneration through selection. These methods can be imposed at thenucleus or elite breeding level where the improved traits would, throughtime, flow to the entire population of animals, or could be implementedat the multiplier or foundation parent level to sort parents into mostgenetically desirable. This invention provides a method for determiningthe optimum male and female parent to maximize the genetic components ofdominance and epistasis thus maximizing heterosis and hybrid vigor inthe market animals.

In another aspect, the present invention provides a bovine subjectresulting from the selection and breeding aspect or the cloning aspectof the invention, discussed above.

In another aspect, the present invention provides a method of trackingmeat of a bovine subject. The method includes identifying nucleotideoccurrences for a series of genetic markers of the bovine subject,identifying the nucleotide occurrences for the series of genetic markersfor a meat sample, and determining whether the nucleotide occurrences ofthe bovine subject are the same as the nucleotide occurrences of themeat sample. In this method identical nucleotide occurrences indicatethat the meat sample is from the bovine subject. The tracking methodprovides, for example, a method for historical and epidemiologicaltracking the location of an animal from embryo to birth through itsgrowth period, to the feedlot and harvest and finally the retail productafter the it has reached the consumer.

The series of genetic markers can be a series of single nucleotidepolymorphisms (SNPs). The method can further include comparing theresults of the above determination with a determination of whether themeat is from the bovine subject made using another tracking method. Inthis embodiment, the present invention provides quality controlinformation that improves the accuracy of tracking the source of meat bya single method alone.

The nucleotide occurrence data for the bovine subject can be stored in acomputer readable form, such as a database. Therefore, in one example,an initial nucleotide occurrence determination can be made for theseries of genetic markers for a young bovine subject and stored in adatabase along with information identifying the bovine subject. Then,after meat from the bovine subject is obtained, possibly months or yearsafter the initial nucleotide occurrence determination, and before and/orafter the meat is shipped to a customer such as, for example, awholesale distributor, a sample can be obtained from the meat andnucleotide occurrence information determined using methods discussedherein. The database can then be queried using a user interface asdiscussed herein, with the nucleotide occurrence data from the meatsample to identify the bovine subject.

A series of markers or a series of SNPs as used herein, can include aseries of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 75, 100, 150, 200, 250, 500, 1000, 2000, 2500, 5000, or 6000markers, for example.

In another aspect, the present invention provides a method fordiagnosing a health condition of a bovine subject. The method includesdrawing an inference regarding a trait of the bovine subject for thehealth condition, from a nucleic acid sample of the subject. Theinference is drawn by identifying, in the nucleic acid sample, at leastone nucleotide occurrence of a single nucleotide polymorphism (SNP),wherein the nucleotide occurrence is associated with the trait.

The nucleotide occurrence of at least 2 SNPs can be determined. The atleast 2 SNPs can form a haplotype, wherein the method identifies ahaplotype allele that is associated with the trait. The method caninclude identifying a diploid pair of haplotype alleles for one or morehaplotypes.

The health condition for this aspect of the invention, is resistance todisease or infection, susceptibility to infection with and shedding ofpathogens such as E. Coli, salmonella, listeria, prion diseases, andother organisms potentially pathogenic to humans, regulation of immunestatus and response to antigens, susceptibility to bloat, liver abscessor the buller syndrome, previous exposure to infection or parasites, orhealth of respiratory and digestive tissues.

The present invention in another aspect provides a method for inferringa trait of a bovine subject from a nucleic acid sample of the subject,that includes identifying, in the nucleic acid sample, at least onenucleotide occurrence of a single nucleotide polymorphism (SNP). Thenucleotide occurrence is associated with the trait, thereby allowing aninference of the trait.

These embodiments of the invention are based, in part, on adetermination that single nucleotide polymorphisms (SNPs), includinghaploid or diploid SNPs, and haplotype alleles, including haploid ordiploid haplotype alleles, allow an inference to be drawn as to thetrait of a subject, particularly a bovine subject.

Accordingly, methods of the invention can involve determining thenucleotide occurrence of at least 2, 3, 4, 5, 10, 20, 30, 40, 50, etc.SNPs. The SNPs can form all or part of a haplotype, wherein the methodcan identify a haplotype allele that is associated with the trait.Furthermore, the method can include identifying a diploid pair ofhaplotype alleles.

In another embodiment, the present invention provides a method foridentifying a bovine genetic marker that influences at least one traitby analyzing bovine genetic markers of a genome-wide genetic marker mapfor association with the trait. The genetic marker can be a singlenucleotide polymorphism (SNP), or can be at least two SNPs thatinfluence the trait. Because the method can identify at least two SNPs,and in some embodiments, many SNPs, the method can identify not onlyadditive genetic components, but non-additive genetic components such asdominance (i.e. dominating trait of an allele of one gene over an alleleof a another gene) and epistasis (i.e. interaction between genes atdifferent loci). Furthermore, the method can uncover pleiotropic effectsof SNP alleles (i.e. SNP alleles or haplotypes effects on many differenttraits), because many traits can be analyzed for their association withmany SNPs using methods disclosed herein.

In one aspect, expression products of genes near the at least twoidentified genetic markers are analyzed, to determine whether theexpression products interact. In certain aspects, at least 2 SNPs areidentified for inferring the genetic potential of a bovine animal forone, two, or more traits. At least 2 of the single nucleotidepolymorphisms are located on different chromosomes. Furthermore, atleast 2 of the single nucleotide polymorphisms can be separated by atleast 10,000 base pairs on the bovine genome. In certain examples, atleast 2 of the single nucleotide polymorphisms occur in different genes.

Accordingly, the present invention provides methods for identifyinggenes, chromosomal regions and SNP markers in bovine animals thataccount for a large proportion of the additive and non-additive geneticvariation observed for any trait that has a genetic component. Themethods and systems of the present invention utilize informationregarding genetic diversity among cattle, particularly single nucleotidepolymorphisms (SNPs), and the effect of nucleotide occurrences of SNPson economically important traits.

The present invention provides methods to allow the simultaneousdiscovery of any and all SNP markers that associate with one or moretraits in one or more regions throughout the entire genome. Furthermore,the present invention provides methods for utilization of the predictivediagnostic to determine the genetic potential of an animal to expressany targeted trait(s). The genetic potential of a bovine animal toexpress multiple economically important traits, termed the molecularbreeding and selection value, is utilized to enhance the efficiency andaccuracy of breeding, sorting and cloning of animals.

The present invention provides methods for developing a high-densitygenetic map of the bovine genome based on single nucleotide polymorphic(SNP) markers. The high-density genetic map is created through a wholegenome sequence of the bovine genome using the shotgun sequencingapproach. Shotgun sequencing is performed with several different bovineindividuals that represent different breed types. Upon whole genomeassembly of the sequenced fragments all sequence variants are identifiedand cataloged. Sequence variants that differ by a single nucleotidebecome candidate SNP markers for the high-density map. The relativeposition of each candidate SNP within the bovine genome is determined byusing the assembled human genome as scaffolding. Candidate SNPs arechosen based on their locations so that the map is evenly distributedacross the bovine genome. The invention includes methods for creating anevenly distributed genetic SNP map where the average genetic distancebetween any two adjacent markers is 0.5 cM (i.e. 500,000 nucleotides).

Furthermore, in one embodiment, the present invention provides methodsfor utilizing a high-density genetic SNP map in the performance of wholegenome association studies in bovine animals and the identification ofstatistically significant associations between specific SNPs andtargeted traits. The invention provides methods for inferring the effectof the associated SNP on the target trait through allele frequencydifferences in the SNP. Furthermore, the invention provides methods foridentifying all SNPs that are adjacent to or in close proximity to theassociated SNP and ascertaining the effect these SNPs have on the targettrait, as disclosed in more detail hereinbelow.

The invention provides methods for pooling DNA samples from bovineindividuals that represent high and low phenotypic extremes for theexpression of a target trait in a population of bovine animals. Thetarget trait can be any trait that has a genetic component and wherephenotypic differences for the trait can be measured in bovine animals,for example marbling, tenderness, fat thickness, yield, daily gain, ormeat quality grade.

The invention provides methods for identifying all genomic regions, andany SNP or set of SNPs contained in these regions, that effect theexpression of a target trait. For example, an inference can be drawnregarding a beef characteristic such as marbling or red meat yield andallele frequency differences in one or more SNPs.

The methods infer the discovery of one or more, and in some cases, allSNPs that show association to a target trait and therefore, account fora large proportion of the genetic variation observed in the expressionof the trait in a population of bovine animals. The methods allowidentification of SNPs that account for additive as well as non-additivegenetic variation, such as dominance and epistasis, observed in theexpression of the trait.

The methods infer the discovery of any and all SNPs that showassociation to one or more target traits. Furthermore, whereby certaintraits have either positive or negative correlations to each other, themethods allow identification of all SNPs that enhance or uncouple thecorrelation. For example, the presence of external fat on beef carcassesis highly correlated with marbling or intra-muscular fat. External fatis an undesirable trait that causes discounts in beef carcasses, whereasmarbling is a desirable trait that results in premiums. The presentinvention provides methods for the identification of all SNPs that mayuncouple the correlation between external fat and marbling, for example.

In another aspect, the present invention provides a method fordeveloping a predictive diagnostic through the identification of one ormore, and in certain aspects all SNPs that are associated with multipletraits having economic significance in bovines, and any and all SNPsthat affect any single trait that are located throughout the entirebovine genome. The methods of the invention result in the development ofa predictive diagnostic system for determining the genetic potential ofindividual animals for any trait that has a genetic component.

In another aspect, the present invention provides a method for utilizinga predictive diagnostic to determine the genetic potential of a bovineanimal for multiple traits located across multiple genomic regions. Thegenetic potential determination for the expression of multiple traits ina bovine animal is referred to as the molecular breeding and selectionvalue. The present invention provides methods for using the molecularbreeding and selection value to enhance efficiencies and accuracy ofbreeding, sorting, purchasing and cloning of individual animals.

Accordingly, nucleotide occurrences can be determined for essentiallyall, or all of the SNPs of a high-density, whole genome SNP map. Thisapproach has the advantage over traditional approaches in that since itencompasses the whole genome, it identifies potential interactions ofgene products expressed from genes located anywhere on the genomewithout requiring preexisting knowledge regarding a possible interactionbetween the gene products. An example of a high-density, whole genomeSNP map is a map of at least about 1 SNP per 10,000 kb, at least 1 SNPper 500 kb or about 10 SNPs per 500 kb, or at least about 25 SNPs ormore per 500 kb. Definitions of densities of markers may change acrossthe genome and are determined by the degree of linkage disequilibriumfrom marker to marker.

In another embodiment of the invention, a method is provided foridentifying SNPs that are associated with a trait by using theassociated SNPs disclosed herein. The method is based on the fact thatother markers in close proximity to the associated SNP marker will alsoassociate with the trait because markers in linkage disequilibrium withthe associated SNP marker will also be in linkage disequilibrium withthe gene(s) influencing the trait. SNPs in linkage disequilibrium can beused in lieu of determining a SNP or mutation to predict the presence orabsence of a phenotypic trait or contributor to a phenotypic trait.Accordingly, in certain embodiments, the present invention provides amethod for identifying a SNP associated with a trait, that includesidentifying a test SNP that is in disequilibrium with a SNPcorresponding to position 300 of SEQ ID NOS:19473 to 21982.

As illustrated in the Examples section, it has been determined thatdisequilibrium exists across the region of 500,000 bp from theassociated SNP in each direction. Other markers within this 500,000 bpregion will also be in disequilibrium with the associated SNP and withthe trait of interest, and can be used to infer associations with thetrait of interest. Genomic segments containing the markers can beadjacent to the associated SNP marker or contained within a separateisland of sequence distant from the associated SNP.

Genetic markers within 500,000 bp of the associated SNPs disclosedherein in Tables 1A and 1B (position 300 of SEQ ID NOS:19473 to 21982),can be discovered by a number of different methods known in the art. Inone aspect of the invention, bovine sequence that is within 500,000 bpof the associated SNP can be used to identify new DNA markers. Thissequence can be created from whole-genome shotgun sequencing,BAC-sequencing, or sequence generated from comparative maps. The bovinesequence can be used to develop bovine specific sequencing primers.These primers can be used to sequence at least 2 individual bovineanimals and the alignments from these sequences can be used to identifySNP markers and microsatellite markers.

New markers can also be discovered using heterologous sequences fromother mammalian species. Degenerative primers are developed from regionsof known homology among species and used in PCR. Amplification productsare sequenced and used to develop bovine specific primers.

These new markers can be genotyped in pools of animals or individualanimals representing the high and low ends of the phenotypicdistribution for the trait to determine association between the newmarker(s) and the trait. Markers with a significantly different allelefrequency in the high and low groups are also in disequilibrium with thetrait.

Accordingly, in another embodiment, the present invention provides amethod for identifying a bovine single nucleotide polymorphism (SNP)associated with a trait that includes identifying a test SNP in a targetregion of a bovine genome, wherein the target region is less than orequal to about 500,000 nucleotides from a SNP position corresponding toposition 300 of one of SEQ ID NOS:19473 to 21982, and identifying anassociation of the test SNP to the trait. In certain aspects, the targetregion consists of at least 20 contiguous nucleotides of SEQ IDNOS:24493 to 64886. In other aspects, the target region consists of atleast 20 contiguous nucleotides of SEQ ID NOS:19473 to 21982.

In certain aspects, the test SNP is located less than or equal to about500,000, 400,000, 300,000, 250,000, 200,000, 100,000, 50,000, 25,000,10,000, 5,000, 1,000, or 100 nucleotides from a position correspondingto position 300 of at least one of SEQ ID NOS:19473 to 21982. The testSNP is expected to be associated with the same trait as a SNP thatcorresponds to position 300 of SEQ ID NOS:19473 to 21982 that is locatedless than or equal to about 500,000 nucleotides from the test SNP, asdiscussed further herein.

The trait can be any bovine trait as discussed herein. For example, thetrait can be marbling, tenderness, quality grade, muscle content, fatthickness, feed efficiency, red meat yield, average daily weight gain,disease resistance, disease susceptibility, feed intake, proteincontent, bone content, maintenance energy requirement, mature size,amino acid profile, fatty acid profile, milk production, susceptibilityto the buller syndrome, stress susceptibility and response, temperament,digestive capacity, production of calpain, caplastatin and myostatin,pattern of fat deposition, ribeye area, fertility, ovulation rate,conception rate, fertility, susceptibility to infection with or sheddingof pathogens. In certain specific examples, the trait is fat thickness,retail yield, tenderness, marbling, or average daily gain.

In another embodiment, the present invention provides a method foridentifying a bovine gene associated with a trait, by identifying anopen reading frame present in a target region of the bovine genome,wherein the target region is located on the bovine genome less than orequal to about 500,000 nucleotides of a single nucleotide polymorphism(SNP) corresponding to position 300 of any one of SEQ ID NOS:19473 to21982, and analyzing the open reading frame to determine whether itaffects the trait.

In certain aspects, the target region is located less than or equal toabout 500,000, 400,000, 300,000, 250,000, 200,000, 100,000, 50,000,25,000, 10,000, 5,000, 1,000, 100, or 50 nucleotides from a singlenucleotide polymorphism (SNP) corresponding to position 300 of any oneof SEQ ID NOS:19473 to 21982.

It will be recognized that a variety of methods can be used to determinewhether the open reading frame affects a trait. For example, biochemicalmethods can be performed to determine a biochemical function for theproduct of the open gene product. The biochemical function can becompared to known biochemical functions related to the trait.Furthermore, the open reading frame can be mutated and the affects ofthe mutations on a target trait can be analyzed.

In another embodiment the present invention provides a method foridentifying a target bovine polynucleotide affecting a trait, thatincludes providing a polynucleotide derived from a bovine subject, orsequence information thereof; and determining whether the polynucleotideis at least 90% identical to a SNP-containing polynucleotide. Thedetermination can be carried out by comparing the polynucleotide or thesequence information to a polynucleotide consisting essentially of:

-   -   a) a polynucleotide according to any one of SEQ ED NOS:19473 to        21982;    -   b) a contiguous fragment of a polynucleotide according to any        one of SEQ ID NOS:24493 to 64886 that is at least 300        nucleotides in length and that comprises a single nucleotide        polymorphism corresponding to position 300 of one of SEQ ID        NOS:19473 to 21982, wherein the polymorphism is associated with        the trait; or    -   c) a complement of a) or b).

If a polynucleotide is identified as at least 90% identical to theSNP-containing polynucleotide, the bovine polynucleotide is a targetpolynucleotide for the trait.

In certain aspects, the polynucleotide is derived from a bovine subjectthat includes bovine genomic sequences. In another aspect, the presentinvention provides an isolated polynucleotide identified according tothis method.

The invention, in another aspect includes methods for creating a highdensity bovine SNP map. The SNP markers and their surrounding sequenceare compared to model organisms, for example human and mouse genomes,where the complete genomic sequence is known and syntenic regionsidentified. The model organism map may serve as a template for ensuringcomplete coverage of the animal genome. The finished map has markersspaced in such a way to maximize the amount of linkage disequilibrium ina specific genetic region.

This map is used to mark all regions of the chromosomes, in a singleexperiment, utilizing thousands of experimental animals in anassociation study, to correlate genomic regions with complex and simpletraits. These associations can be further analyzed to unravel complexinteractions among genomic regions that contribute to the targeted traitor other traits, epistatic genetic interactions and pleiotropy. Theinvention of regional high density maps can also be used to identifytargeted regions of chromosomes that influence traits.

Accordingly, in embodiments where SNPs that affect the same trait areidentified that are located in different genes, the method can furtherinclude analyzing expression products of genes near the identified SNPs,to determine whether the expression products interact. As such, thepresent invention provides methods to detect epistatic geneticinteractions. Laboratory methods are well known in the art fordetermining whether gene products interact.

The method can be useful for inferring a beef characteristic from anucleic acid sample of the subject animal (i.e., the trait is acharacteristic of beef). Beef characteristics that can be inferred bymethods of the present invention include, for example, overall quality,marbling, red meat yield, tenderness, and the like. Accordingly, thepresent invention provides methods for identifying live cattle that haveor that lack the genetic potential to produce beef that is well-marbled.Such information could be used by the cattle producer to channel calvesinto particular feeding regimens and to meet the requirements ofspecific marketing programs. Such information could also be used toidentify cattle that are genetically superior candidates for breedingand/or cloning. Such information could also be used to identify cattlethat are genetically inferior candidates to be screened out of abreeding or cloning program.

Where the trait is overall quality, the method can infer an overallaverage USDA quality grade for beef obtained from the non-bovinelivestock subject. For example, the quality grades can include one ofthe current eight USDA quality grades (i.e., from highest to lowest,prime, choice, select, standard, commercial, utility, cutter andcanner). Alternatively, the method can infer the best or the worstquality grade expected for beef obtained from the non-bovine livestocksubject. Additionally, as indicated above, the trait can be acharacteristic used to classify beef, such as color, texture, firmness,and marbling, a term which is used to describe the relative amount anddistribution of intramuscular fat of the beef. Well-marbled andwell-distributed beef from steers and heifers, i.e., beef that containssubstantial amounts of intramuscular fat relative to muscle, isclassified as prime or choice; whereas, beef that is not marbled isclassified as select. Beef that is classified as prime or choice,typically, is sold at higher prices than beef that is classified intolower quality grades.

Where the trait is red meat yield, the method can predict the total andpercentage of edible product from a harvested animal. For example, Yieldgrade is assigned to an animal from an estimate of its back fatthickness, kidney-pelvic-heart fat, ribeye area and carcass weight.Grades range from 1 to 5, with 1 being the greatest retail product yieldassignment. This method can predict the traits for red meat yield andquality grade and tailor a feeding, management and harvesting program tooptimize the value of the animal.

The methods of the present invention that infer a trait can be used inplace of present methods used to determine the trait, or can be used tofurther substantiate a classification of beef using present methods(e.g., visual inspection of a region between the 12th and 13th rib of abeef carcass by a certified USDA grader). Where the trait is tenderness,for example, methods of the present invention can infer from a sample ofa bovine subject, such as a live bovine subject, whether beef, if cookedproperly, would be tender. The method can be used in place of currentpost-mortem taste tests or shear force methods, or can be used toimprove the accuracy of determinations made by these traditionalmethods.

In aspects of the present invention directed at identifying a bovinegenetic marker that influences a trait, present methods for determininga trait, such as a characteristic of beef, can be used in the methods toidentify an association between a genetic marker, typically at least oneSNP or haplotype, with a trait. For example, DNA samples from bovinesubjects can be obtained, and nucleotide occurrences for at least oneSNP in the DNA samples can be determined. Traditional methods can beused to determine the trait. For example, visual inspection of a regionbetween the 12th and 13th rib of a beef carcass can be performed todetermine the quality grade of meat obtained from the bovine subjectwhose nucleotide occurrences are identified. As will be understood,statistical methods can then be used to identify associations betweenthe nucleotide occurrences and the trait. Accordingly, methods of thepresent invention enables a correlation between carcass value andgenetic variation, so as to help identify superior genetic types forfuture breeding or cloning and management purposes, and to identifymanagement practices that will maximize the value of the arrival in themarket.

In another aspect, the present invention provides a method foridentifying a bovine gene associated with a trait that includesidentifying a bovine single nucleotide polymorphism (SNP) thatinfluences a trait, by analyzing a genome-wide bovine SNP map forassociation with the trait, wherein the SNP is found on a target regionof a bovine chromosome. Genes present on the target region are thenidentified. The presence of a gene on the target region of the bovinechromosome indicates that the gene is a candidate gene for associationwith the trait. The candidate gene can then be analyzed using methodsknown in the art to determine whether it is associated with the trait.

In another aspect, the present invention provides a method foridentifying a breed of a bovine subject. The method includes identifyinga nucleotide occurrence of a bovine single nucleotide polymorphism (SNP)from a nucleic acid sample of the subject, wherein the nucleotideoccurrence is associated with the breed of the subject. The methodtypically includes identifying nucleotide occurrences of at least twoSNPs from the nucleic acid sample, wherein the nucleotide occurrencesare associated with the breed of the subject.

In another aspect, the present invention provides a system fordetermining the nucleotide occurrences at a population of bovine singlenucleotide polymorphisms (SNPs). The system typically includes ahybridization medium and/or substrate that includes at least twooligonucleotides of the present invention, or oligonucleotides used inthe methods of the present invention. For example, a solid support canbe provided, to which a series of oligonucleotides can be directly orindirectly attached. In another aspect, a homogeneous assay is includedin the system. In another aspect, a microfluidic device is included inthe system. The hybridization medium or substrates are used to determinethe nucleotide occurrence of bovine SNPs that are associated with atrait.

Accordingly, the oligonucleotides are used to determine the nucleotideoccurrence of bovine SNPs that are associated with a trait. Thedetermination can be made by selecting oligonucleotides that bind at ornear a genomic location of each SNP of the series of bovine SNPs. Thesystem of the present invention typically includes a reagent handlingmechanism that can be used to apply a reagent, typically a liquid, tothe solid support. The binding of an oligonucleotide of the series ofoligonucleotides to a polynucleotide isolated from a genome can beaffected by the nucleotide occurrence of the SNP. The system can includea mechanism effective for moving a solid support and a detectionmechanism. The detection method detects binding or tagging of theoligonucleotides.

Medium to high-throughput systems for analyzing SNPs, known in the artsuch as the SNPStream® UHT Genotyping System (Beckman/Coulter,Fullerton, Calif.) (Boyce-Jacino and Goelet Patents), the Mass Array™system (Sequenom, San Diego, Calif.) (Storm, N. et al. (2002) Methods inMolecular Biology. 212: 241-262.), the BeadArray™ SNP genotyping systemavailable from Illumina (San Diego, Calif.) (Oliphant, A., et al. (June2002) (supplement to Biotechniques), and TaqMan™ (Applied Biosystems,Foster City, Calif.) can be used with the present invention. However,the present invention provides a medium to high-throughput system thatis designed to detect nucleotide occurrences of bovine SNPs, or a seriesof bovine SNPs that can make up a series of haplotypes. Therefore, asindicated above the system includes a solid support or other method towhich a series of oligonucleotides can be associated that are used todetermine a nucleotide occurrence of a SNP for a series of bovine SNPsthat are associated with a trait. The system can further include adetection mechanism for detecting binding of the series ofoligonucleotides to the series of SNPs. Such detection mechanisms areknown in the art.

The system can be a microfluidic device. Numerous microfluidic devicesare known that include solid supports with microchannels (See e.g., U.S.Pat. Nos. 5,304,487, 5,110745, 5,681,484, and 5,593,838).

The SNP detection systems of the present invention are designed todetermine nucleotide occurrences of one SNP or a series of SNPs. Thesystems can determine nucleotide occurrences of an entire genome-widehigh-density SNP map.

Numerous methods are known in the art for determining the nucleotideoccurrence for a particular SNP in a sample. Such methods can utilizeone or more oligonucleotide probes or primers, including, for example,an amplification primer pair that selectively hybridizes to a targetpolynucleotide, which corresponds to one or more bovine SNP positions.Oligonucleotide probes useful in practicing a method of the inventioncan include, for example, an oligonucleotide that is complementary toand spans a portion of the target polynucleotide, including the positionof the SNP, wherein the presence of a specific nucleotide at theposition (i.e., the SNP) is detected by the presence or absence ofselective hybridization of the probe. Such a method can further includecontacting the target polynucleotide and hybridized oligonucleotide withan endonuclease, and detecting the presence or absence of a cleavageproduct of the probe, depending on whether the nucleotide occurrence atthe SNP site is complementary to the corresponding nucleotide of theprobe.

An oligonucleotide ligation assay (Grossman, P. D. et al. (1994) NucleicAcids Research 22:4527-4534) also can be used to identify a nucleotideoccurrence at a polymorphic position, wherein a pair of probes thatselectively hybridize upstream and adjacent to and downstream andadjacent to the site of the SNP, and wherein one of the probes includesa terminal nucleotide complementary to a nucleotide occurrence of theSNP. Where the terminal nucleotide of the probe is complementary to thenucleotide occurrence, selective hybridization includes the terminalnucleotide such that, in the presence of a ligase, the upstream anddownstream oligonucleotides are ligated. As such, the presence orabsence of a ligation product is indicative of the nucleotide occurrenceat the SNP site. An example of this type of assay is the SNPlex System(Applied Biosystems, Foster City, Calif.).

An oligonucleotide also can be useful as a primer, for example, for aprimer extension reaction, wherein the product (or absence of a product)of the extension reaction is indicative of the nucleotide occurrence. Inaddition, a primer pair useful for amplifying a portion of the targetpolynucleotide including the SNP site can be useful, wherein theamplification product is examined to determine the nucleotide occurrenceat the SNP site. Particularly useful methods include those that arereadily adaptable to a high throughput format, to a multiplex format, orto both. The primer extension or amplification product can be detecteddirectly or indirectly and/or can be sequenced using various methodsknown in the art. Amplification products which span a SNP locus can besequenced using traditional sequence methodologies (e.g., the“dideoxy-mediated chain termination method,” also known as the “SangerMethod” (Sanger, F., et al., J. Molec. Biol. 94:441 (1975); Prober etal. Science 238:336-340 (1987)) and the “chemical degradation method,”also known as the “Maxam-Gilbert method” (Maxam, A. M., et al., Proc.Natl. Acad. Sci. (U.S.A.) 74:560 (1977)), both references hereinincorporated by reference) to determine the nucleotide occurrence at theSNP locus.

Methods of the invention can identify nucleotide occurrences at SNPsusing genome-wide sequencing or “microsequencing” methods. Whole-genomesequencing of individuals identifies all SNP genotypes in a singleanalysis. Microsequencing methods determine the identity of only asingle nucleotide at a “predetermined” site. Such methods haveparticular utility in determining the presence and identity ofpolymorphisms in a target polynucleotide. Such microsequencing methods,as well as other methods for determining the nucleotide occurrence at aSNP locus are discussed in Boyce-Jacino, et al., U.S. Pat. No.6,294,336, incorporated herein by reference, and summarized herein.

Microsequencing methods include the Genetic Bit™ Analysis methoddisclosed by Goelet, P. et al. (WO 92/15712, herein incorporated byreference). Additional, primer-guided, nucleotide incorporationprocedures for assaying polymorphic sites in DNA have also beendescribed (Kornher, J. S. et al, Nucleic Acids Res. 17:7779-7784 (1989);Sokolov, B. P., Nucleic Acids Res. 18:3671 (1990); Syvanen, A.- C., etal., Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl.Acad. Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al, Hum.Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992);Nyren, P. et al., Anal. Biochem. 208:171-175 (1993); and Wallace,WO89/10414). These methods differ from Genetic Bit™ Analysis in thatthey all rely on the incorporation of labeled deoxynucleotides todiscriminate between bases at a polymorphic site. In such a format,since the signal is proportional to the number of deoxynucleotidesincorporated, polymorphisms that occur in runs of the same nucleotidecan result in signals that are proportional to the length of the run(Syvanen, A.- C., et al. Amer. J. Hum. Genet. (1993) 52:46-59 Otherformats for microsequencing include Pyrosequencing (Pyrosequencing AB,Uppsala, Sweden, Alderborn et al (2000) Genome Res. 10:1249-1258).

Alternative microsequencing methods have been provided by Mundy, C. R.(U.S. Pat. No. 4,656,127) and Cohen, D. et al (French Patent 2,650,840;PCT Appln. No. WO91/02087) which discusses a solution-based method fordetermining the identity of the nucleotide of a polymorphic site. As inthe Mundy method of U.S. Pat. No. 4,656,127, a primer is employed thatis complementary to allelic sequences immediately 3′-to a polymorphicsite.

In response to the difficulties encountered in employing gelelectrophoresis to analyze sequences, alternative methods formicrosequencing have been developed. Macevicz (U.S. Pat. No. 5,002,867),for example, describes a method for determining nucleic acid sequencevia hybridization with multiple mixtures of oligonucleotide probes. Inaccordance with such method, the sequence of a target polynucleotide isdetermined by permitting the target to sequentially hybridize with setsof probes having an invariant nucleotide at one position, and variantnucleotides at other positions. The Macevicz method determines thenucleotide sequence of the target by hybridizing the target with a setof probes, and then determining the number of sites that at least onemember of the set is capable of hybridizing to the target (i.e., thenumber of “matches”). This procedure is repeated until each member of aset of probes has been tested.

Boyce-Jacino, et al., U.S. Pat. No. 6,294,336 provides a solid phasesequencing method for determining the sequence of nucleic acid molecules(either DNA or RNA) by utilizing a primer that selectively binds apolynucleotide target at a site wherein the SNP is the most 3′nucleotide selectively bound to the target.

The occurrence of a SNP can be determined using denaturing HPLC such asdescribed in Nairz K et al (2002) Proc. Natl. Acad. Sci. (U.S.A.)99:10575-80, and the Transgenomic WAVE® System (Transgenomic, Inc.Omaha, Nebr.).

Oliphant et al. report a method that utilizes BeadArray™ Technology thatcan be used in the methods of the present invention to determine thenucleotide occurrence of a SNP (supplement to Biotechniques, June 2002).Additionally, nucleotide occurrences for SNPs can be determined using aDNAMassARRAY system (SEQUENOM, San Diego, Calif.). This system combinesproprietary SpectroChips™, microfluidics, nanodispensing, biochemistry,and MALDI-TOF MS (matrix-assisted laser desorption ionization time offlight mass spectrometry).

As another example, the nucleotide occurrences of bovine SNPs in asample can be determined using the SNP-IT™ method (Beckman Coulter,Fullerton, Calif.). In general, SNP-IT™ is a 3-step primer extensionreaction. In the first step a target polynucleotide is isolated from asample by hybridization to a capture primer, which provides a firstlevel of specificity. In a second step the capture primer is extendedfrom a terminating nucleotide triphosphate at the target SNP site, whichprovides a second level of specificity. In a third step, the extendednucleotide trisphosphate can be detected using a variety of knownformats, including: direct fluorescence, indirect fluorescence, anindirect colorimetric assay, mass spectrometry, fluorescencepolarization, etc. Reactions can be processed in 384 well format in anautomated format using a SNPstream™ instrument (Beckman Coulter,Fullerton, Calif.). Reactions can also be analyzed by binding to Luminexbiospheres (Luminex Corporation, Austin, Tex., Cai. H. (2000) Genomics66(2):135-43.). Other formats for SNP detection include TaqMan™ (AppliedBiosystems, Foster City, Calif.), Rolling circle (Hatch et al (1999)Genet. Anal. 15: 35-40; and Qi et al (2001) Nucleic Acids Research Vol.29 e 116), fluorescence polarization (Chen, X., et al. (1999) GenomeResearch 9:492-498), SNaPShot (Applied Biosystems, Foster City, Calif.;and Makridakis, N. M. et al. (2001) Biotechniques 31:1374-80),oligo-ligation assay (Grossman, P. D., et al. (1994) Nucleic AcidsResearch 22:4527-4534), locked nucleic acids (LNATM, Link, TechnologiesLTD, Lanarkshire, Scotland, EP patent 1013661, U.S. Pat. No. 6,268,490),Invader Assay (Aclara Biosciences, Wilkinson, D. (1999) The Scientist13:16), padlock probes (Nilsson et al. Science (1994), 265: 2085),Sequence-tagged molecular inversion probes (similar to padlock probes)from ParAllele Bioscience (South San Francisco, Calif.; Hardenbol, P. etal. (2003) Nature Biotechnology 21:673-678), Molecular Beacons (Marras,S. A. et al. (1999 Genet Anal. 14:151-156), the READIT™ SNP GenotypingSystem from Promega (Madison, Wis.) (Rhodes R. B. et al. (2001) MolDiagn. 6:55-61), Dynamic Allele-Specific Hybridization (DASH) (Prince,J. A. et al. (2001) Genome Research 11:152-162), the Qbead™ system(quantum dot encoded microspheres conjugated to allele-specificoligonucleotides) (Xu H. et al. (2003) Nucleic Acids Research 31:e43),Scorpion primers (similar to molecular beacons except unimolecular)(Thelwell, N. et al. (2000) Nucleic Acids Research 28:3752-3761), andMagiprobe (a novel fluorescence quenching-based oligonucleotide probecarrying a fluorophore and an intercalator) (Yamane A. (2002) NucleicAcids Research 30:e97). In addition, Rao, K. V. N. et al. ((2003)Nucleic Acids Research. 31:e66), recently reported a microsphere-basedgenotyping assay that detects SNPs directly from human genomic DNA. Theassay involves a structure-specific cleavage reaction, which generatesfluorescent signal on the surface of microspheres, followed by flowcytometry of the microspheres. With a slightly different twist on theSequenom technology (MALDI), Sauer et al. ((2003) Nucleic Acids Research31:e63) generate charge-tagged DNA (post PCR and primer extension),using a photocleavable linker.

Accordingly, using the methods described above, the bovine haplotypeallele or the nucleotide occurrence of a bovine SNP can be identifiedusing an amplification reaction, a primer extension reaction, or animmunoassay. The bovine haplotype allele or bovine SNP can also beidentified by contacting polynucleotides in the sample orpolynucleotides derived from the sample, with a specific binding pairmember that selectively hybridizes to a polynucleotide region comprisingthe bovine SNP, under conditions wherein the binding pair memberspecifically binds at or near the bovine SNP. The specific binding pairmember can be an antibody or a polynucleotide.

The nucleotide occurrence of a SNP can be identified by othermethodologies as well as those discussed above. For example, theidentification can use microarray technology, which can be performedwith or without PCR, or sequencing methods such as mass spectrometry,scanning electron microscopy, or methods in which a polynucleotide flowspast a sorting device that can detect the sequence of thepolynucleotide. The occurrence of a SNP can be identified usingelectrochemical detection devices such as the eSensor™ DNA detectionsystem (Motorola, Inc., Yu, C. J. (2001) J. Am Chem. Soc.123:11155-11161). Other formats include melting curve analysis usingfluorescently labeled hybridization probes, or intercalating dyes(Lohmann, S. (2000) Biochemica 4, 23-28, Herrmann, M. (2000) ClinicalChemistry 46: 425).

The SNP detection systems of the present invention typically utilizeselective hybridization. As used herein, the term “selectivehybridization” or “selectively hybridize,” refers to hybridization undermoderately stringent or highly stringent conditions such that anucleotide sequence preferentially associates with a selected nucleotidesequence over unrelated nucleotide sequences to a large enough extent tobe useful in identifying a nucleotide occurrence of a SNP. It will berecognized that some amount of non-specific hybridization isunavoidable, but is acceptable provide that hybridization to a targetnucleotide sequence is sufficiently selective such that it can bedistinguished over the non-specific cross-hybridization, for example, atleast about 2-fold more selective, generally at least about 3-fold moreselective, usually at least about 5-fold more selective, andparticularly at least about 10-fold more selective, as determined, forexample, by an amount of labeled oligonucleotide that binds to targetnucleic acid molecule as compared to a nucleic acid molecule other thanthe target molecule, particularly a substantially similar (i.e.,homologous) nucleic acid molecule other than the target nucleic acidmolecule. Conditions that allow for selective hybridization can bedetermined empirically, or can be estimated based, for example, on therelative GC:AT content of the hybridizing oligonucleotide and thesequence to which it is to hybridize, the length of the hybridizingoligonucleotide, and the number, if any, of mismatches between theoligonucleotide and sequence to which it is to hybridize (see, forexample, Sambrook et al., “Molecular Cloning: A laboratory manual (ColdSpring Harbor Laboratory Press 1989)).

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42EC (moderate stringency conditions); and0.1×SSC at about 68EC (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

The term “polynucleotide” is used broadly herein to mean a sequence ofdeoxyribonucleotides or ribonucleotides that are linked together by aphosphodiester bond. For convenience, the term “oligonucleotide” is usedherein to refer to a polynucleotide that is used as a primer or a probe.Generally, an oligonucleotide useful as a probe or primer thatselectively hybridizes to a selected nucleotide sequence is at leastabout 15 nucleotides in length, usually at least about 18 nucleotides,and particularly about 21 nucleotides or more in length.

A polynucleotide can be RNA or can be DNA, which can be a gene or aportion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence,or the like, and can be single stranded or double stranded, as well as aDNA/RNA hybrid. In various embodiments, a polynucleotide, including anoligonucleotide (e.g., a probe or a primer) can contain nucleoside ornucleotide analogs, or a backbone bond other than a phosphodiester bond.In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotideor oligonucleotide also can contain nucleotide analogs, includingnon-naturally occurring synthetic nucleotides or modified naturallyoccurring nucleotides. Such nucleotide analogs are well known in the artand commercially available, as are polynucleotides containing suchnucleotide analogs (Lin et al., Nucleic Acids Research (1994)22:5220-5234 Jellinek et al., Biochemistry (1995) 34:11363-11372;Pagratis et al., Nature Biotechnol. (1997) 15:68-73, each of which isincorporated herein by reference). Primers and probes can also becomprised of peptide nucleic acids (PNA) (Nielsen P E and Egholm M.(1999) Curr. Issues Mol. Biol. 1:89-104).

The covalent bond linking the nucleotides of a polynucleotide generallyis a phosphodiester bond. However, the covalent bond also can be any ofnumerous other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like bond or any other bond known to those in the art asuseful for linking nucleotides to produce synthetic polynucleotides(see, for example, Tam et al., Nucl. Acids Res. (1994) 22:977-986, Eckerand Crooke, BioTechnology (1995) 13:351360, each of which isincorporated herein by reference). The incorporation of non-naturallyoccurring nucleotide analogs or bonds linking the nucleotides or analogscan be particularly useful where the polynucleotide is to be exposed toan environment that can contain a nucleolytic activity, including, forexample, a tissue culture medium or upon administration to a livingsubject, since the modified polynucleotides can be less susceptible todegradation.

A polynucleotide or oligonucleotide comprising naturally occurringnucleotides and phosphodiester bonds can be chemically synthesized orcan be produced using recombinant DNA methods, using an appropriatepolynucleotide as a template. In comparison, a polynucleotide oroligonucleotide comprising nucleotide analogs or covalent bonds otherthan phosphodiester bonds generally are chemically synthesized, althoughan enzyme such as T7 polymerase can incorporate certain types ofnucleotide analogs into a polynucleotide and, therefore, can be used toproduce such a polynucleotide recombinantly from an appropriate template(Jellinek et al., supra, 1995). Thus, the term polynucleotide as usedherein includes naturally occurring nucleic acid molecules, which can beisolated from a cell, as well as synthetic molecules, which can beprepared, for example, by methods of chemical synthesis or by enzymaticmethods such as by the polymerase chain reaction (PCR).

A method of the identifying a SNP also can be performed using a specificbinding pair member. As used herein, the term “specific binding pairmember” refers to a molecule that specifically binds or selectivelyhybridizes to another member of a specific binding pair. Specificbinding pair member include, for example, probes, primers,polynucleotides, antibodies, etc. For example, a specific binding pairmember includes a primer or a probe that selectively hybridizes to atarget polynucleotide that includes a SNP loci, or that hybridizes to anamplification product generated using the target polynucleotide as atemplate.

As used herein, the term “specific interaction,” or “specifically binds”or the like means that two molecules form a complex that is relativelystable under physiologic conditions. The term is used herein inreference to various interactions, including, for example, theinteraction of an antibody that binds a polynucleotide that includes aSNP site; or the interaction of an antibody that binds a polypeptidethat includes an amino acid that is encoded by a codon that includes aSNP site. According to methods of the invention, an antibody canselectively bind to a polypeptide that includes a particular amino acidencoded by a codon that includes a SNP site. Alternatively, an antibodymay preferentially bind a particular modified nucleotide that isincorporated into a SNP site for only certain nucleotide occurrences atthe SNP site, for example using a primer extension assay.

A specific interaction can be characterized by a dissociation constantof at least about 1×10⁻⁶M, generally at least about 1×10⁻⁷ M, usually atleast about 1×10⁻⁸M, and particularly at least about 1×10⁻⁹M or 1×10⁻¹⁰M or greater. A specific interaction generally is stable underphysiological conditions, including, for example, conditions that occurin a living individual such as a human or other vertebrate orinvertebrate, as well as conditions that occur in a cell culture such asused for maintaining mammalian cells or cells from another vertebrateorganism or an invertebrate organism. Methods for determining whethertwo molecules interact specifically are well known and include, forexample, equilibrium dialysis, surface plasmon resonance, and the like.

The invention also relates to kits, which can be used, for example, toperform a method of the invention. Thus, in one embodiment, theinvention provides a kit for identifying nucleotide occurrences orhaplotype alleles of bovine SNPs. Such a kit can contain, for example,an oligonucleotide probe, primer, or primer pair, or combinationsthereof. Such oligonucleotides being useful, for example, to identify aSNP or haplotype allele as disclosed herein; or can contain one or morepolynucleotides corresponding to a portion of a bovine gene containingone or more nucleotide occurrences associated with a bovine trait, suchpolynucleotide being useful, for example, as a standard (control) thatcan be examined in parallel with a test sample. In addition, a kit ofthe invention can contain, for example, reagents for performing a methodof the invention, including, for example, one or more detectable labels,which can be used to label a probe or primer or can be incorporated intoa product generated using the probe or primer (e.g., an amplificationproduct); one or more polymerases, which can be useful for a method thatincludes a primer extension or amplification procedure, or other enzymeor enzymes (e.g., a ligase or an endonuclease), which can be useful forperforming an oligonucleotide ligation assay or a mismatch cleavageassay; and/or one or more buffers or other reagents that are necessaryto or can facilitate performing a method of the invention. The primersor probes can be included in a kit in a labeled form, for example with alabel such as biotin or an antibody.

In one embodiment, a kit of the invention provides a plurality ofoligonucleotides of the invention, including one or more oligonucleotideprobes or one or more primers, including forward and/or reverse primers,or a combination of such probes and primers or primer pairs. Such a kitalso can contain probes and/or primers that conveniently allow a methodof the invention to be performed in a multiplex format.

The kit can also include instructions for using the probes or primers todetermine a nucleotide occurrence of at least one bovine SNPs.

In another aspect, the present invention provides a computer system thatincludes a database having records containing information regarding aseries of bovine single nucleotide polymorphisms (SNPs), and a userinterface allowing a user to input nucleotide occurrences of the seriesof bovine SNPs for a bovine subject. The user interface can be used toquery the database and display results of the query. The database caninclude records representing some or all of the SNP of a bovine SNP map,such as a high-density bovine SNP map. The database can also includeinformation regarding haplotypes and haplotype alleles from the SNPs.Furthermore, the database can include information regarding traitsand/or traits that are associated with some or all of the SNPs and/orhaplotypes. In these embodiments the computer system can be used, forexample, for any of the aspects of the invention that infer a trait of abovine subject.

The computer system of the present invention can be a stand-alonecomputer, a conventional network system including a client/serverenvironment and one or more database servers, and/or a handheld device.A number of conventional network systems, including a local area network(LAN) or a wide area network (WAN), are known in the art. Additionally,client/server environments, database servers, and networks are welldocumented in the technical, trade, and patent literature. For example,the database server can run on an operating system such as UNIX, runninga relational database management system, a World Wide Web application,and a World Wide Web Server. When the computer system is a handhelddevice it can be a personal digital assistant (PDA) or another type ofhandheld device, of which many are known.

Typically, the database of the computer system of the present inventionincludes information regarding the location and nucleotide occurrencesof bovine SNPs. Information regarding genomic location of the SNP can beprovided for example by including sequence information of consecutivesequences surrounding the SNP, that only 1 part of the genome provides100% match, or by providing a position number of the SNP with respect toan available sequence entry, such as a Genbank sequence entry, or asequence entry for a private database, or a commercially licenseddatabase of DNA sequences. The database can also include informationregarding nucleotide occurrences of SNPs, since as discussed hereintypically nucleotide occurrences of less than all four nucleotides occurfor a SNP.

The database can include other information regarding SNPs or haplotypessuch as information regarding frequency of occurrence in a bovinepopulation. Furthermore, the database can be divided into multipleparts, one for storing sequences and the others for storing informationregarding the sequences. The database may contain records representingadditional information about a SNP, for example information identifyingthe gene in which a SNP is found, or nucleotide occurrence frequencyinformation, or characteristics of the library or clone which generatedthe DNA sequence, or the relationship of the sequence surrounding theSNP to similar DNA sequences in other species.

The parts of the database of the present invention can be flat filedatabases or relational databases or object-oriented databases. Theparts of the database can be internal databases, or external databasesthat are accessible to users. An internal database is a databasemaintained as a private database, typically maintained behind afirewall, by an enterprise. An external database is located outside aninternal database, and is typically maintained by a different entitythan an internal database. A number of external public biologicalsequence databases, particularly SNP databases, are available and can beused with the current invention. For example, the dbSNP databaseavailable from the National Center for Biological Information (NCBI),part of the National Library of Medicine, can be used with the currentinvention to provide comparative genomic information to assist inidentifying bovine SNPs.

In another aspect, the current invention provides a population ofinformation regarding bovine SNPs and haplotypes. The population ofinformation can include an identification of traits associated with theSNPs and haplotypes. The population of information is typically includedwithin a database, and can be identified using the methods of thecurrent invention. The population of sequences can be a subpopulation ofa larger database, that contains only SNPs and haplotypes related to aparticular trait. For example, the subpopulation can be identified in atable of a relational database. A population of information can includeall of the SNPs and/or haplotypes of a genome-wide SNP map.

In addition to the database discussed above, the computer system of thepresent invention includes a user interface capable of receiving entryof nucleotide occurrence information regarding at least one SNP. Theinterface can be a graphic user interface where entries and selectionsare made using a series of menus, dialog boxes, and/or selectablebuttons, for example. The interface typically takes a user through aseries of screens beginning with a main screen. The user interface caninclude links that a user may select to access additional informationrelating a bovine SNP map.

The function of the computer system of the present invention thatcarries out the trait inference methods typically includes a processingunit that executes a computer program product, itself representinganother aspect of the invention, that includes a computer-readableprogram code embodied on a computer-usable medium and present in amemory function connected to the processing unit. The memory functioncan be ROM or RAM.

The computer program product, itself another aspect of the invention, isread and executed by the processing unit of the computer system of thepresent invention, and includes a computer-readable program codeembodied on a computer-usable medium. The computer-readable program coderelates to a plurality of sequence records stored in a database. Thesequence records can contain information regarding the relationshipbetween nucleotide occurrences of a series of bovine single nucleotidepolymorphisms (SNPs) and a trait of one or more traits. The computerprogram product can include computer-readable program code for providinga user interface capable of allowing a user to input nucleotideoccurrences of the series of bovine SNPs for a bovine subject, locatingdata corresponding to the entered query information, and displaying thedata corresponding to the entered query. Data corresponding to theentered query information is typically located by querying a database asdescribed above.

In another embodiment of the present invention, the computer system andcomputer program products are used to perform bioeconomic valuationsused to perform methods described herein, such as methods for estimatingthe value of a bovine subject or meat that will be obtained therefrom.

The following examples are intended to illustrate but not limit theinvention.

Example 1 Generation of a High-Density Bovine Genetic SNP Map

This example illustrates the generation of a high density bovine geneticSNP map created through a whole genome sequencing of the bovine genomeusing the shotgun sequencing approach. This approach was selected toprovide hundreds of thousands of SNP markers, as described by Venter, J.C, et al., (Science 291:1304-1351 (2001), in order to perform awhole-genome association study with adequate density of markers toensure discovery of markers in disequilibrium with mutations influencingthe targeted traits.

Shotgun sequencing was performed with four different bovine individualsthat represented different breed types. The shotgun sequencing wasperformed according to the methods of Venter, J. C, et al., (Science291:1304-1351 (2001)). By this method, random fragments of bovinesequence were generated and size selected to 2.5 and 10 kb. Thesefragments of bovine DNA were inserted into a sequencing vector to createhigh quality plasmid libraries suitable for high throughput sequencing.

Shotgun sequencing was performed with four different bovine subjectsthat represented several different breed types: Angus, Limousin, Brahmanand Simmental. Upon whole genome assembly of the sequenced fragments,contigs were formed from consensus sequence, and sequence variants wereidentified and cataloged. 786,777 sequence variants that differed by asingle nucleotide became candidate SNP markers for the high-density SNPmap. The relative position of each candidate SNP within the bovinegenome was determined using the assembled human genome as scaffoldingcreating a candidate map of 242,181 human-mapped markers. Uponpositioning of the SNPs within the genome, individual markers weretested to determine informativeness within the cattle population using210 animals representing diverse breeds (Angus, Charolais, Limousin,Hereford, Brahman, Simmental and Gelbvieh) and Mendialian segregation(21 trios of parents and progeny). Selected markers were polymorphic inthe majority of the breeds tested. Any markers within a region thatfailed the test were discarded and replaced with another marker in theregion. These markers were also validated against the test population.This process was repeated until a relatively evenly distributed geneticSNP map was obtained, where the average genetic distance between any twoadjacent markers is 0.5 cM.

Example 2 Identification of Bovine SNPs Associated with Tenderness, Fat,Marbling, Yield, and/or Daily Gain

This example illustrates the identification of SNPs from thehigh-density bovine SNP map identified in Example 1, that are associatedwith the traits meat tenderness, fat thickness, marbling, yield, and/ordaily gain.

DNA samples from bovine subjects were obtained by collecting 50 ml ofwhole blood from the 4,791 bovine subjects. 25 ml of whole blood wasused for DNA extraction using standard methods and concentrations of DNAwere calculated using standard fluorimetric methods. Animalsrepresenting less than or equal to the 10th percentile of low numericphenotypic animals (44 individuals) and the 90th percentile and greaterof high phenotypic animals (44 individuals) were identified for eachtrait. The low numeric values were identified as “Low” and the highnumeric values were identified as “High”. DNA samples were pooled frombovine individuals that represent high and low phenotypic extremes forthe expression of a target trait in a population of bovine animals witheach of the 44 animals contributing equally to the pool of DNA. Aseparate “High” and “Low” pool was created for each biological type(English, Continental, and Brahman crosses) by treatment group (Early,Optimum, Late) for each of the five traits resulting in 90 total pools.In addition to the 90 pools listed above, another group was formed basedon animals that were 5 standard deviations above the mean for numerictenderness values. Eleven animals were included in this group ofindividuals and the pool was compared to the other tenderness groupsresulting in a total of 91 pools. Each pool was tested against each ofthe 6189 mapped and validated SNP markers. The SNP detection platformutilized in the experiment was the Beckman Coulter SNP-IT system,utilizing single-base extension of the SNP base. Allele frequency wasestimated for each pool based on the fluorescence intensity of each ofthe two incorporated fluorescent labels corresponding to the SNPalleles. These estimates were adjusted for marker specificcharacteristics and incorporation differences. A test statistic wasdeveloped based on a Chi-square distribution of differences among allelefrequencies of the high minus low pools. These test statistics weresummed across the 9 breed by treatment groups within each traitresulting in Chi-square distribution. SNP markers reaching a thresholdtest statistic of 46.96294 for the trait of tenderness and 21.66599(p<0.01) for the remaining four traits of retail yield, daily gain, fatthickness and marbling were identified as associated SNPs and are listedin Tables 1A and 1B.

The high-density SNP map was used to identify SNPs that are associatedwith a series of bovine traits. The traits included marbling,tenderness, fat thickness, yield, and daily gain. Tables 1A and 1B(filed herewith on a compact disc) provide the identity of SNPs thatassociated with one or more of the traits analyzed. Twenty five hundredand ten associated SNPs were identified for all five traits.

Table 1A provides the following information, from left to right columns:SNP name; a sequence identifier of the sequence listing filed herewith,for an amplicon, wherein the SNP position is position 300 of theamplicon; position of the SNP within the amplicon (i.e. position 300);The nucleotide sequence and SEQ ID NO: for an extension primer capableof priming polynucleotide synthesis across the SNP position; trait(s)that are associated with the SNP; Characteristics of the trait that areassociated with specific nucleotide occurrences at the SNP; Nucleotideoccurrences that have been detected at the SNP position; And thesequence identifier of contig sequences that are located within 500,000nucleotides from the SNP on the bovine genome. Table 1B provides thefollowing information from left to right columns: SNP name; A sequencename of a contig that includes the SNP position, as well as the positionnumbers within the contig for an amplicon that includes the SNP;Position of the SNP within the amplicon (i.e. position 300); Thenucleotide sequence for an extension primer capable of primingpolynucleotide synthesis across the SNP position; trait(s) that areassociated with the SNP; Characteristics of the trait that areassociated with specific nucleotide occurrences at the SNP; Nucleotideoccurrences that have been detected at the SNP position; And thesequence identifier of contig sequences that are located within 500,000nucleotides from the SNP on the bovine genome.

Example 3 Determination of the Distance of Disequilibrium in Cattle

This example utilizes a few of the associated SNPs disclosed in Example2, to identify additional SNPs that are associated with the same traits,using the physical proximity on the genome of the SNPs. Furthermore, theresults are used to calculate a distance of disequilibrium in cattle. Inthis example, “shear force” is used to refer to tenderness, “visionretail yield” is used to refer to retail yield, and “average daily gain”is used to refer to daily gain.

In the past 10 years numerous methods have been developed to identifyalleles associated with phenotypic effects, traits or diseases. Linkagedisequilibrium and measures of linkage disequilibrium have been ofparticular interest for studies of complex traits or diseases (seereviews L. R. Cardon and J. I. Bell, “Association study Designs forComplex Diseases”, Nature Reviews/Genetics 2:91-99 (2001); N. A.Rosenberg and M. Nordborg “Genealogical Trees, Coalescent Theory and theanalysis of Genetic Polymorphisms”, Nature Reviews/Genetics 3:380-390,2002). LD occurs where blocks or regions of neighboring markers areco-inherited from a common ancestor. The degree of LD variesconsiderably throughout the genome and is a function of time,recombination events, mutation rate and population structure. The extentof LD can vary from a few thousand base pairs to several centimorgans.This has been most extensively documented in human studies (K. W. Bromanand J. L. Weber. “Long homozygous chromosomal segments in the CEPHfamilies”. Am. J. Hum. Genet. 65: 1493-1500 (1999); A. G. Clark, K. M.Weiss, D. A. Nickerson, et. al. “Haplotype structure and populationgenetic inferences from nucleotide-sequence variation in humanlipoprotein lipase. Am. J. Hum. Genet. 63:595-612 (1999); D. Reich, M.Cargill, S. Bolk, et al., “Linkage disequilibrium in the human genome”.Nature 411:199-204 (2001); J. Stephens, J. A. Schneider, D. A. Tanguay,et al., “Haplotype variation and linkage disequilibrium in 313 humangenes”. Science 293:489-493 (2001); E. Dawson, G R Abecasis, S.Bumpstead, et al “A first generation linkage disequilibrium map of humanchromosome 22”. Nature 418(6897):544-548 (2002); S B Gabriel, S FSchaffner, H Nguyen, et al. “The structure of haplotype blocks in thehuman genome” Science 296: 225-2229 (2002)). Similar results have beenobserved in other species including cattle (F. Farnir, W. Coppieters, J-J. Arranz, et. al., “Extensive Genome-wide Linkage Disequilibrium inCattle” Genome Research 10:220-227 (2000)). These studies and othershave also shown that a SNP or multiple SNPs associated with a phenotypecan be used as predictive of gene(s) causing differences in traitphenotypes within a region of high LD although they may or may not bethe precise causative gene (as further examples, see also: A. M.Glazier, J H Nadeau and T J Aitman, “Finding Genes that Underlie ComplexTraits” Science 298: 2345-2348 (2002); M. Blumenfield, et al. U.S. Pat.No. 6,531,279; A, Hovnanian, et al., U.S. Pat. Pub. No. 20030190637A1;M. Blumenfield, et al. U.S. Pat. No. 6,528,260; M. R. Hayden, et al.U.S. Pat. No. 6,617,122; C. M. Drysdale, et al. U.S. Pat. No. 6,586,183;M. Galvin, et al., U.S. Pat. No. 6,586,175; L. Bougueleret, et al., U.S.Pat. No. 6,582,909; S. Van Dijk, et al., U.S. Pat. No. 6,558,905. A. E.Anastasio, et al., U.S. Pat. No. 6,521,741). While it has beenestablished that markers can be identified that associate with aspecific trait, and, therefore, become diagnostic for the trait, thedistance that disequilibrium reaches has not been determined in cattlewith a dense marker map. Therefore, an experiment to determine thedisequilibrium distance in cattle was performed using the high-densitySNP map disclosed in Example 1.

The high-density SNP map disclosed in Example 1 was used to identifySNPs that are in physical proximity to a few of the associated SNPsdisclosed in Example 2. Nucleotide occurrences of the SNPs weredetermined using the method disclosed in Example 2. A determination ofwhether on-test SNPs was associated with a trait was performed asdisclosed in Example 2.

As discussed above, the study was performed to verify the assumptionthat markers that are in close physical proximity on the bovine genomewill associate with the same trait(s) because markers in linkagedisequilibrium with the associated SNP marker will also be in linkagedisequilibrium with the mutation(s) influencing the trait.

As indicated in Table 2, SNP3 (MMBT22302) is significantly associatedwith the trait of average daily gain (“ADG” in Table 2). Several SNPswere identified using the high-density SNP map of Example 1 that arelocated at various distances from SNP3 on the bovine genome (Table 2).For example, SNP2 is 466,047 nucleotides from SNP3. Furthermore, SNP5was identified which is 408,732 nucleotides from SNP3. SNP6 wasidentified which is 1.0 million nucleotides from SNP3. Finally, SNP4 wasidentified, which is 308,742 nucleotides.

As illustrated in Table 2, SNPs that were located within 500,000nucleotides of SNP3 also were associated with average daily gain,whereas those that were located greater than 500,000 nucleotides fromSNP3 were not associated with average daily gain. For example, linkagedisequilibrium reaches 466,047 bases to SNP2, but not to SNP1 at 1.5 Mb;linkage disequilibrium reaches to 408,732 bases to SNP5, but not to SNP6at 1.0 Mb. SNP4, which is 308,742 nucleotides from SNP3, was discoveredby sequencing the contig of DNA that maps to this region in 4 differentbreeds of cattle. It is also in disequilibrium with average daily gain.

TABLE 2 Disequilibrium analysis in relation to SNP distance fromMMBT22302. Marker At Position Association Human 300 in SEQ P < .01Chromosome Difference from SNP ID NO Trait Location bp locationMMBT22302 1 MMBT22310 not in patent Not Sign HC16 45425130 1,507,460 2MMBT13976 20291 ADG HC16 46466543 466,047 3 MMBT22302 19666 ADG HC1646932590 4 MMBT09532 21944 ADG HC16 47241332 308,742 5 MMBT09533 19999ADG HC16 47341322 408,732 6 MMBT09535 21078 Not Sign HC16 479582461,025,656

To further analyze linkage disequilibrium, a similar analysis wasperformed using another SNP identified as an associated SNP in Example2. SNP9 (MMBT03905) is significantly associated with vision retail yield(VRY). SNPs 7-8 and 10-12 were identified that are various distancesfrom SNP9 (Table 2). Again, SNPs that were located less than or equal toabout 500,000 nucleotides from the associated SNP, were also associatedwith the trait, whereas those that were present greater than 500,000nucleotides from a known associated SNP, were not associated. Forexample, SNPs 8 and 11 were identified as also being highlysignificantly associated with VRY and are located less than 500,000 bpfrom SNP9. (Table 3). On the other hand, SNPs 7 and 12, which aregreater than 500,000 bp from SNP9, were not associated with the trait.Furthermore, through additional sequencing, SNP10 was discovered andalso found to be in linkage disequilibrium with VRY.

TABLE 3 Disequilibrium analysis in relation to SNP distance fromMMBT3905. Marker At Position Association Human 300 in SEQ P < .01Chromosome Difference from SNP ID NO Trait Location bp locationMMBT03905 7 MMBT12437 not in patent Not Sign HC04 177035705 518426 8MMBT03904 20327 VRY HC04 177201331 352800 9 MMBT03905 19816 VRY HC04177554131 10 MMBT03906 20240 VRY HC04 177900170 346039 11 MMBT0590620045 VRY HC04 178047550 493419 12 MMBT03907 not in patent Not Sign HC04178113631 559500

As indicated in Tables 1A and 1B, SNP16 (MMBT02782) is highlysignificantly associated with shear force (SHF, Table 4). SNPs 14, 15,17 and 18 were identified which are located within 500,000 nucleotidesof SNP16 (Table 4). Once again, all of these SNPs, which are within500,000 nucleotides of an associated SNP, were found to be associatedwith the same trait. That is, SNPs 14, 15, 17, and 18 were all found tobe associated with SHF (Table 4). On the other hand, SNPs 1 and 7, whichare located beyond 1.0 million nucleotides from SNP16, were notassociated with SHF.

TABLE 4 Disequilibrium analysis in relation to SNP distance fromMMBT02782. Marker At Position Association Human 300 in SEQ P < .01Chromosome Difference from SNP ID NO Trait Location bp locationMMBT02782 13 MMBT02777 19767 Not Sign HC04 46401363 1594271 14 MMBT0278120791 SHF HC04 47777758 217876 15 MMBT19460 20790 SHF HC04 47778002217632 16 MMBT02782 20901 SHF HC04 47995634 17 MMBT03688 20765 SHF HC0448379141 383507 18 MMBT02784 20764 SHF HC04 48492482 496848 19 MMBT02786not in patent Not Sign HC04 49190953 1195319

The results of this Example indicate that disequilibrium in cattleexists across the region of 500,000 nucleotides from an associated SNP,in each direction. Therefore, it is expected that when an associated SNPis identified, other markers within this 500,000 bp region will also bein disequilibrium with the associated SNP and with the trait ofinterest, and can be used to infer associations with the trait ofinterest.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1-100. (canceled)
 101. A method of matching a bovine trait-associatedgenotype with a bovine subject comprising identifying in a nucleic acidsample from the bovine subject an occurrence of at least three singlenucleotide polymorphisms (SNPs), wherein the at least three SNPs areassociated with a trait, wherein the at least three SNPs occur in morethan one gene, and sorting the bovine subject based on the associatedtrait.